Chapter G.

Steps 31-40 RNA and the First Bions

Step 31. Nucleotide evolution.
In the descriptions of the origins and evolution of the components of nucleotides and of the nucleotides themselves, some details have been left out. In reference to earlier chemical steps, the Miller-Urey experiments (now confirmed in part by recent discoveries on other celestial objects in the Solar system) and deep Ocean probes of the last century, it seems clear that many molecules, including various organic compounds, including cyanide; oxides of carbon, sulfur, and phosphate; methane and other hydrocarbons; water, salts, and the Ocean; alcohols and carboxylic acids; ammonia and amines derived from it; and amino acids all had formed on Earth by 4.4 billion years ago at the latest. In the Ocean, phosphates would have existed near the geothermal plumes.

White smokers emitting liquid carbon dioxide at the Champagne vent, Northwest Eifuku volcano, in the Marianas Trench Marine National Monument (from Wikimedia Commons, article hydrothermal vent). It is near such hydrothermal vents (as they are better known now) that the author hypothesizes biology began on Earth.

The exact timing of later steps is less clear, but the existence of montmorillonite clay (an aluminum compound) in the vicinity of the geothermal plumes surely implies the formation of ring peptides soon afterward. Some time may have elapsed before enough amino acids of the right composition arose to confer enzymatic functions on these ring peptides, but I would guess that it was surely happening by 4,300 millads ago (4.3 Ga), allowing 100 millads (100 million years) to accomplish that stage. In the meantime, sugars, likely including ribose, would have appeared, since they do so in open space, where conditions for formation are far less favorable. Thus, phosphorus tetroxide ions and ribose would soon after be forming, increasing the likelihood of the formation of the organic bases, also derivable from other sources. Most likely, some pyrimidine nucleotides existed earlier, perhaps well before 4,200 millads ago (4.2 Ga).

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Because of their simpler and smaller structure, pyrimidines likely formed before purines. Uracil and cytodine, among the pyrimidines, most likely appeared first, at slightly different times. Thymine is not found in the simplest or in the least changed descendants of the earliest types of biota, so it probably appeared later. As noted in step 29, the existence of some few nucleotides most likely provided a better atmosphere for Ocean geochemistry to produce both larger numbers of individual types of existing nucleotides and perhaps even further types of organic heterocyclic bases and their nucleotides. So the purines may have started forming by 4,250 millads ago (4.25 billion years ago), more or less. The first organic bases described so far, and thus the nucleotides containing them, most likely did not originally have their present formulae but, once formed, continued to change. If they influenced the formation of more somewhat like them, any accidental change in formula that increased that likelihood would be a type surviving better than other types, while a formula leading to fewer (or no) further examples would decline. The influence may only have made a few percent difference per generation, or one percent, or a fraction of a percent, but molecules form, change, break up rapidly, so numerous generations change in a day, or sometimes even in hours. At that rate, even very slight influences can have a significant cumulative effect over the years. The original formation of organic bases as well as their later changes (evolution), and their binding into nucleotides, were surely influenced and assisted by their chemical and physical environment, including temperature, chemical neighbors, and their own effects on that environment and other molecules. Perhaps influential as well were mineral or ionic metal catalysts and other convenient surfaces and some peptide-ring enzymes. I have not seen reports of experiments on this last point, but it appears highly probable, though the details remain to be established. Very likely one or more, most probably several such influences, played a part in the next step. By that time there may already have been the purines adenine and guanine. If step 32 started before purines existed, step 32 must have pushed them into existence quickly.

Step 32. RNA and the first bion.

a. RNA chains After combining some organic bases and phosphate ions into nucleotides by binding them to the ends of ribose molecules, it happens that nucleotides tend to join each other as monomers in their own turn, creating a polymer chain of these called ribonucleic acid, abbreviated as RNA. RNA is made of four kinds of nucleotide and may have a wide range of lengths. The shortest of which have read has three nucleotides but that does not necessarily mean that two-nucleotide RNA does not exist. At the other extreme, RNA chains are known to reach at least hundreds of nucleotides in length, at least about 500. b. Outside assistance What has been said in the previous step about chemical and physical environmental influences and of the influence of metallic and other catalysts, peptide-ring enzymes, and of phosphate ions with their high chemical energy and nucleotides themselves on the origin, survival, and evolution of the nucleotides and their predecessors and parts also naturally applies to the formation of RNA chains. It is likely that some peptide-ring enzyme effect was important

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in helping this step to occur. That was why this work mentioned amino acids in the first place. Both phosphate ions and nucleotides that were not part of the new RNA helped provide the necessary energy boost to those nucleotides that did join to make the chaining more frequent. These helping chemicals would only have been numerous enough near the geothermal vents and plumes in the deep Ocean, where temperatures were higher than in other parts of the Ocean or on land. c. Composition and structure The four nucleotides in the type of RNA that appears to have been first are the purines, adenine and guanine, and the pyrimidines, uracil and cytosine. (We shall come back to inosine and thymine later.) The reader will recall that (1) a ring nitrogen atom of an organic base binds that base into the nucleotide by binding to a ring carbon (between the ring oxygen and another ring carbon) of the ribose. (2) On the ring-carbon atom, on the opposite side of the ring oxygen of the ribose, a non-ring carbon is bound with its two hydrogen companions, i.e., a methyl group. (3) To that methyl carbon a phosphate ion binds, completing the nucleotide.

Nucleotide structure (from Wikimedia Commons): composed of a nitrogenous base, a five-carbon sugar, and one or more phosphate groups. Each of the purines and pyrimidines, here shown in blue, can form the base, which is in yellow in the diagram of the nucleotide itself.

So the ribose holds the nucleotide together with bonds on both sides. Ribose also tends to attach itself (and thus its whole nucleotide) to the phosphate ion of another nucleotide. So the RNA chain is built, because the phosphate binds on two sides, like an amino acid, but the ribose binds on three sides, as shown in the diagram. The whole chain becomes an acid, even though its contains weak bases, because phosphate is strongly acidic (note its potential hydrogen ions). The four nucleotides that constitute RNA can assemble in any order and in any proportions.38 A laboratory experiment created a kind of RNA chain composed entirely of one kind of nucleotide, so such an RNA is possible.39 Each position in the chain can be any one of the four nucleotides, making a very large number of possible chains only several nucleotides long. There is no way to be sure what the precise arrangement was in the earliest individual chains, so from our point of view we can regard the arrangements as random.
38

The author is proposing a version of the RNA world hypothesis, also proposed by others. See, for example, Atkins, John F., Raymond F. Gesteland, and Thomas Cech. 2006. The RNA world: the nature of modern RNA suggests a prebiotic RNA world. Plainview, NY: Cold Spring Harbor Laboratory Press. Carl

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Woese was apparently the first to publish on this hypothesis (1967. The Genetic Code. New York: Harper and Row). Others include F.H.C. Crick (1968. The origin of the genetic code, in Journal of Molecular Biology 38: 367-379) and L.E. Orgel (1968. Evolution of the genetic apparatus, in Journal of Molecular Biology 38: 381-393). 39 The genetic material for the earliest bions, if not RNA, may have been simpler nucleic acid polymers: Orgel, Leslie. 2000. Perspective: Origin of Life: A Simpler Nucleic Acid, in Science 290 (5495): 1306-1307, doi:10.1126/science.290.5495.1306, referring to Schoening, K.-U., P. Scholz, S. Guntha, X. Wu, R. Krishnamurthy, and A. Eschenmoser. 2000. Chemical Etiology of Nucleic Acid Structure: The Threofuranosyl (32) Oligonucleotide System, in Science 290 (5495): 1347-1351, doi:10.1126/science.290.5495.1347. This might be the lab experiment the author refers to. See also Lincoln, Tracey A. and Gerald F. Joyce. 2009. Self-Sustained Replication of an RNA Enzyme, in Science 323 (5918): 1229-1232. Doi:10.1126/science.1167856.

There can be a big difference between two chains with different numbers and arrangements of the various nucleotides. Different ones would have more or less chance of surviving for any meaningful period of time. Different ones would have different qualities of many kinds. The first RNA chains were likely fairly short, but varied in length, survival, and other qualities. d. Characteristics, qualities, and potentialities of RNA RNA, relatively simple as it is, still has a surprising range of potential capabilities, even though most of them were not displayed at first. We cannot yet call the first of them, assembled as described above, yet bions, although the title of this step is the first bion, because they were not made by a nucleic-acid template process, although they constituted the first available such templates. We shall come back to that. (1) Among their potential capabilities, RNA chains can have enzymatic qualities, affecting their own survival and behavior or that of other molecules. This capability is in one way broader than that of the previously existing, ring-peptide enzymes, which can only affect other molecules, not themselves. This is an important potential because it enables them to cause chemical interactions, especially assemblies of both complementary copies of themselves and other molecules, either to get started sooner or to move faster than they otherwise would in the existing conditions, a crucial need for most bions. To distinguish this function from the enzyme abilities of peptides, this capability in an RNA chain is called a ribozyme. (2) A second useful potential is that of folding themselves into various relatively firm states and practical shapes. One advantage of this ability is that it tends to make the RNA chain more stable and less likely to be disrupted by mishaps such as high temperature, disruptive and violent movements of the Oceans waters, or attack by other molecules. A folded shape may tend to protect some parts of the chain from such disruptions or chemical attacks. A second advantage is that a specific shape may improve enzyme function by fitting the molecule to be affected by the enzyme action more precisely and placing the chains own most active part adjacent to the part of the target molecule to be affected. Other advantages will come up in later steps. (3) The third and most important capability, not possessed by many molecules, is that an RNA chain can act as a template to induce the formation of a new complementary copy of itself. How can this happen? We will come back to that.

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This combination of potential abilities lets an RNA chain or strand affect other molecules in ways useful to the RNA, so that it is not entirely at the mercy of the passing mishaps and lost opportunities of an ordinary molecule; so that it survives longer than without such abilities; and possibly even so that it indirectly copies itself, increasing the number, variety, and other capabilities of its progeny. The molecule itself, of course, has no awareness of these facts or any others and might not care if it did, but it therefore has the potential to initiate biology. e. Initiating biology The first RNA strands mostly did not fulfill any of these potentialities but broke up in some mishap or another without making any contribution to future biology. These strands probably never existed in large numbers and did not at first reproduce either very much or very accurately. They were, however, assisted in the reproduction of complementary copies of themselves by their own nature and by the energy provided by phosphate ions and separate nucleotides, outside the strand. So a few did make such copies and those were, in fact, true bions made by biological template replication. As both the original strands and their copies varied from each other in the precise numbers and arrangement of each of the nucleotides, they therefore had slightly different actual (not just potential) abilities and these differences made some more and others less likely to survive and reproduce. Both biology and biological evolution had begun and the outcome of each generation affected the range of qualities and success in the next generation. It is time to give these strands a name and look at their first realized ability. Since they were the first bions, an appropriate name would mean just that: protobions. That first realized ability was the one capability most basic to all biota: reproduction. Because they were so different from the other kinds of biology that have existed on Earth, they deserve to have a separate category of the highest classification in that field, an empire. So they were in the protobion empire. Within that largest category, those that we have met first were and are the least able to do this one thing they could (and can) do, namely, reproduce. The modern forms of these already have a name, so we should use it for them: viroid. So we have now met the viroid kingdom within the protobion empire. These all consist of a single strand of RNA. How exactly did they reproduce? Besdies the outside help already mentioned, their reproduction can be described as follows. f. Matching nucleotides

We already are aware that the nucleotides within a single strand of RNA may come in any proportions and order. This strand then becomes a pattern that has, because of its shape and electrical attractions, a preferred matching pattern. Because of their relative sizes, the matching nucleotides must normally add up to the same number of rings to make the two strands fit together. That number has to be three. So a pyrimidine (with one ring) must match up to a purine (with two rings) and vice versa. This is the first and firmest rule. The pattern is the reason we call this a template process. Mechanical model makers are familiar with templates. A template for them is usually a flat piece of material with the exact profile of the object to be made cut out of it, so the two can be fitted together to see whether the shape is correct. A lathe operator turns the piece of raw material on the lathe until it looks close to the right shape, then takes the flat template and places it near or on that object to see how well they fit together, like a hand in a glove. The hand in a glove is not exactly like the

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inside of the glove because the one is a solid object while the other is a hole, but they can match up. They are exact opposite, one solid where the other is an empty space; they are complementary. That is like what the template process in biology is not making the new strand exactly like the old one, not a copy, but its match or complement. If the process is precise and accurate enough, then when the second generation reproduces, its offspring will be exactly like the grandparent in the viroid kingdom of the protobion empire. To make that work well, there is a second rule for matching. That one, not followed quite as firmly as the first, sets a specific nucleotide that should pair with or match up to another one, more precisely than just pyrimidine to purine. That matching is precise today but it may not always have been so. Even now, the process slips a bit from time to time. The proper or usual matches in viroids are: Adenine matches uracil (and vice versa); and Guanine matches cytosine (and vice versa).

Step 33. Protobion empire, second kingdom: single-stranded RNA with copy-initiation ribozyme.
This outline has mentioned a few of the many potentialities that RNA chains or strands have as protobions. But protobions of the first kingdom, the viroids, mostly did not achieve many of those potentials, remaining merely passive molecular strands of variable lengths that might, in ideal circumstances, have served as templates for the formation of new viroids. The copying method was not completely accurate, with considerable error (a few to several percent). As a result, this kingdom probably never became large. Yet it did manage to produce one step toward greater autonomy, survivability, and copying efficiency. At least one viroid match (i.e. offspring) became endowed, in the inaccurate template-matching process, with a sort of internal ribozyme-like starting ability. This both started the template-matching process and indentified where in the strand to being the process. This single step had a huge influence on future protobions. The bion that represented that step, the offspring mentioned, naturally produced far more offspring than any others before and started a second kingdom with this new actual (not merely potential) ability. We can call that new kingdom by the title used in the heading of this step, shortening it to single-stranded starter RNA. We can abbreviate it further to ssBRNA. Here the ss means single-stranded. The RNA, of course, means the same as before. The B means simply bion, because in these early bions, the RNA constituted either the whole bion or, later, its defining characteristic. The need for this particular letter arises from the fact that various special functions of RNA are customarily distinguished from each other by inserting a letter designating that special function: tRNA (transfer RNA), rRNA (ribosomal RNA), tmRNA (transfermessenger RNA), etc. The letters for these specialties are customarily lower case, but the B is too basic and encompassing to deserve anything less than a super-special designation (i.e., the capital letter). This innovation quickly made the second kingdom grow far bigger than the first had been, as it remains today. By increasing this population, the innovation also increased the likelihood of further simple innovations and those began to come. All the potentials of the first kingdom began to be phased in by small steps as realities, as will be summarized in the next step. Biology was in motion, diversity increased, and our ancestors were on their way. It may be hard to imagine oneself or even ones ancestors as only an RNA strand of nucleotide molecules held together internally and among themselves by tiny electrical attractions. But the most important accomplishments have not been building the tallest or scariest things but rather the most basic things. When did this step happen? We cannot yet determine exactly. It could have been before 44 hundred millads ago (4.4 billion years ago or 4.4Ga) or possibly as late as four billion years ago. I would guess perhaps 44 hundred millads. Yet all that has been accomplished in all the steps in this volume so far has become part of all biology on Earth and has lasted in us all to this day. Even now, specializations of ssRNA perform a crucial role in nearly every process that makes us, and keeps us, alive. Even the starter for our reproduction and that of each of our cells is a short bit of ssRNA.

Step 34. Matching, looping, and the start of specializing within ssBRNA.

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We are aware that complementary nucleotides tend to match up, adenine to uracil and guanine to cytosine. That enables the protobion form of reproduction, where complementary nucleotides may collect, one at a time as they become available, on the existing template protobion, until they form a continuous row, where an adequate energy source, catalyst, or enzyme can enable them to assemble into a second strand. At this stage, their separation into specific protobions is still difficult, but may also have been catalyzed or just helped by arrivals of still more nucleotides that would tend to dislodge one strand from the other. The same template-matching process also permits complementary nucleotides in two parts of the same protobion to attract each other. If it is just one nucleotide attracting another, and especially if they are very close together, no such matching attachment will occur because the nearby nucleotides will repel too greatly. But if several nucleotides in one part of the strand exactly match a complementary group equally long in another part, they usually do match up and form a sort of double-stranded section. This remains part of the single basic strand, but the matching parts line themselves up together side by side and the non-matching parts between bend over as part of the single strand, forming a single-stranded loop at the end of the doublestranded stem section, extending from the side of the main single strand (see figure below).

A hairpin loop from a pre-mRNA blue represents the ribose-phosphate backbone, with green nucleobases (from Wikimedia Commons) a single strand of RNA that has looped back on itself.

Such a stem-loop structure can sometimes form with as few as three nucleotides in the loop, but such a tight loop strains the structure, so normally the loop will be larger, i.e., have more nucleotides (five or so). This special structure can help to serve any one or more of several useful functions. First, it generally tends to stabilize the single main strand, making it stronger and more likely to survive longer. Second, the stem-loop can participate in giving the whole ssBRNA a particular shape, which may either tend to protect it from certain physical or chemical dangers, or increase the enzymatic (or rather ribozymatic) influence of the molecule, or part of it. Third, a stem-loop may naturally arise from the location of the same or matching internal sequences of nucleotides running in both directions. Such an arrangement could have survival value in the influence it has going both ways. That later could help when genes begin to segregate, but that probably did not happen at first. Even such a structure formed because of this would also have the first advantage mentioned and, rarely, the second. The fourth important, potential function of a stem-loop structure would be to increase the sensitivity of a sensor. Suppose a section of the ssBRNA has a small series of nucleotides

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that happens to have a modest attraction for a molecule whose presence would be helpful to the whole ssBRNA. We shall call that series S. If a set of series that are complementary to each (C and C) are added on either side of S, like bookends, then C and C will tend to line up together as a double-stranded stem, and the sensor series S will form a loop, extending out from the main strand. This loop will be more likely to sense an approaching or passing molecule than the same section when flat with the main strand, or even slightly surrounded by larger, smoother curves in the main strand such as those caused by water movements. It is a bit like putting a lookout on a tower instead of having him watch from a plain. Such structures do seem to be formed in this kind of bion. It is also imaginable that something like the fourth function could have an alternative function. Instead of seizing a useful molecule, like a complementary nucleotide, to join as part of a matching strand in reproduction or to collect an amino acid, the stem-loop might repel a dangerous molecule, or react by causing a portion of the main loop to change shape to avoid the danger, or might sense excessive water motion or temperature and cause the whole ssBRNA to change shape to gain greater stability to ride out the crisis. I am not aware, however, of any report of such sensors being found on actual protobions.

Step 35. Precursors of tRNA.

Precursors of a class of specialized RNA types seem to have arisen from the stem-loop structure mentioned as the fourth important function above. The original formed by accident with no initial function. It would, however, have served a stabilizing function, but it seems also to have become a sort of sensor, detecting (and presumably reacting to) an amino acid. Initially, the detection-reaction may not have been helpful, but further accidental40 changes in internal nucleotide sequence from reproduction errors (and preserved by elimination of less able, alternative versions) would have resulted in better sensitivity, more precise identification (if that made a difference in survival rate), and more effective response.
40

I do not really believe that individual particles have accidents. They do what they do for reasons, because of their previous rate and direction of motion, their behavioral tendencies, and the similar behavior of other particles in the vicinity. Yet what they do may be considered accidents from the point of view of their effects on bions, because neither the bion at issue, here a protobion, nor we can predict the outcome. (Authors footnote)

Early in its development, the structure likely detected and reacted only to some broad, general category, perhaps to any hydrogen ion (and hence acid) or perhaps to the carboxyl group, identifying an organic acid, or perhaps to any amine. Then further lapse of time and pressure of competition would lead to a more precise narrowing of the target molecule, say, to an amino acid in general (both carboxyl and amine in the same molecule), or later to a particular category of amino acids, such as the polar ones (ionic) or the non-polar ones (neutral). Later, the development of the incipient or pre-tRNA gradually increased in specificity to specify a particular smaller group. Also, during all that time, new similar sensors were arising, permitting greater control of circumstances important to the survival of the bion and the actual number of amino acids affecting the ssBRNA may have grown. Ultimately, those sensors became fairly accurate and effective at their general tasks, differentiated a little in multiple version on the same ssBRNA, or different ones, and became the precursors of tRNA. The modern tRNAs are separate molecules that do, with help, identify and attach to particular amino acids. But they must have been doing so for a long time because,

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before they became so specific, genetic control of peptides (and later of proteins) would have been too poor for the rise of the genetic code or the prokaryotes, both of which are very old. Examination of modern tRNA molecules reveals some interesting pertinent characteristics. (1) Even today, they are still all stem-loop structures. (2) They are, however, somewhat larger than the usual stem-loop structure. (3) Further, they each have three end loops of the usual type and a theoretical structure that can most easily be diagrammed as a sort of central loop or vestibule, less well defined. (4) All of these tRNA molecules have three main double-stranded subsidiary stems connecting the end loops to the central or vestibule area, and the main stem is also still present, connecting to the vestibule. (5) The whole thing is still a single ssRNA molecule, despite its double-stranded parts. (6) Some tRNA molecules also have yet another lump or truncated stem, of varying lengths. See figure below.

Secondary cloverleaf structure of tRNA from yeast (from Wikimedia Commons, article Transfer RNA). Capital letters represent the bases (adenine, cytosine, guanine, and uracil). Some of these bases are modified (m indicating by methylation): Greek letter represents pseudouridine (derived from uracil), D indicates dihydrouridine. The green numeral 5 is the beginning and the 3 at the top the end of the molecule. See Wikipedia article on tRNA for further details.

Furthermore, this structure today (7) is a single, separate molecule, no longer just a deformation in a long, bionic, single strand. Because of that, the (8) main stem, which looks like the original stem, is no longer connected to a bion. (9) Instead, one side of the main stem extends beyond the other side, thus forming a short, single-stranded, end piece. (10) This single-stranded piece at the end tends to attract and hold a particular amino acid, different for each type of tRNA molecule. If this step originally led to the rest of the parent bion, how did this change come about, and, more importantly, why did it survive? Finally, (11) the loop farthest from the main stem starting and ending points of the single strand has a set of three consecutive nucleotides. (12) These match three consecutive complementary nucleotides on main bion RNA (so far, we have called it BRNA, as it is in protobions). That permits the tRNA molecule to match and join its loop end with a BRNA (temporarily) and with an amino acid at the open, other end.

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What does this structure imply about the history? We have no reports of intermediate stages actually being found. They were too long ago and too ephemeral. All trace of them quickly disappears when their bion dies. The overall shape of tRNA (which is not a bion because it was not the result of template reproduction) implies that the main stem was originally part of a bion, stood up (or down, or outward; orientation would not matter in the Ocean) from the main BRNA strand, and the farthest loop end was adapted to capturing nucleotides, probably to assist reproduction (or replication, as virologists term it). Why did the structure become so big? Because its function was important to creating descendants, so sticking out farther extended its reach and improved its effectiveness. But why the two or three extra side stem-loop structures? Partly, no doubt, because they helped stiffen the whole structure and reduce the likelihood of its missing the prey because of leaning or falling over. In addition, at one time, the nucleotides in those extra loops may have helped catch a passing nucleotide. Besides that, current form suggests that the whole structure (1) stand up from the BRNA but also (2) bend over to provide the outermost loop to another structure for part of the needed process. (The next step discusses what that other structure may have become.) A further consideration involves the evident reversal of the role of the main stem. If it started as a stem, other changes in the abilities of its original bion may have required addition of another function. As tends to happen in organic chemistry and in biology generally, an effective structure tends to be recreated numerous times and some of these become adapted to slightly differing functions. (I call this multiplication and diversification. It happens among bions and among their parts.) As genes began to develop to influence amino acid behavior, to create helpful peptides, thereby escaping the limitation of waiting for just the right peptide enzyme to appear accidentally, the need for a sensor to attract amino acids would arise. The slightest change in that direction, if it had the least useful effect, would have survived and thus survival pressure would lead to improvements. Such a sensor-attractor may have formed on the main strand of the protobion and, at some point, may have been relatively close to one of the nucleotide catchers, especially as the number of special structures along the bion increased, crowding them closer together. Perhaps the newer amino acid senor stood nearby or came loose at one end by a mutation, leaving it only attached through the tRNA stem, and later the intervening nucleotides were eliminated, combining both sensors in one structure. Perhaps again the extra two arms (side stem-loops) permitted a stepwise roll of the whole structure from one attachment to another, again through complementarity of the ends of the arms to the main bion, enabling either end to function. However it happened, and the intervening steps all had to be useful at the time in surviving or reproducing, ultimately it produced a change in the function of the structure, and finally in its relationship to the bion. The bion was enables to collect amino acids and create its own peptides, gaining better control of its destiny. Some of these peptides became enzymes of the common type, and some structural, as we shall see later. To do this, though, some other things had to change as well. Today, all biota except the protobions possess many tRNA molecules in almost every cell. (The surviving protobions no longer need them to be parts of themselves but they still use those of other bions.) In the beginning, protobions had to have and use tRNA molecules to help them influence their environment, increase their chances of survival and reproduction, achieve the other forms of progress that they had to do to increase their numbers in a world of limited resources, and, incidentally, they led to the rest of biology. Those tRNA molecules (small molecules with 70 to 80 varying nucleotides) today exist in dozens of types, each adapted to capturing just one kind of amino acid and to rejoining the

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linear RNA strand that specifies (indirectly) which amino acid is needed in order to build each peptide needed. That strand, four thousand millads ago (4 Ga) was the ssBRNA. (Today, it is usually a similar single strand specially made and used only for that specific purpose, then broken and recycled immediately afterward. We call these modern strands messenger RNA, abbreviated mRNA.) That rejoining of the main strand happens because three nucleotides in the middle of the tRNA loop farthest from the stem complement three nucleotides in the mRNA strand (that is, it now happens in the mRNA but it was in the bion in the beginning). Just as in the reproduction or replication of the strand itself, this rejoining is template-matched, cytosine with guanine, uracil with adenine.

Step 36. Pre-rRNA.

Another set of changes must have become underway at least during the later part of tRNA development and evolution, and closely interwoven with it. Its origin is less clear, but its timing and outcome (up to now) are evident. It included three major specialized RNA molecules (and, in our time and part of biology, a fourth smaller one, separated from one of the other three original molecules). These may have arisen separately, in different protobions, or together in one line of descent. Their present counterparts may have been parts of larger bions, structures built and loosely held by bions, cast off, or attached to different parts of bions. These parts have different but related functions and ultimately ended up wrapped together (accompanied now by some proteins) in a single, small, protobion-sized, compact, roundish, biological object called a ribosome (from Greek sma, body). This object plays a crucial part in all bions outside the protobion empire but is like, and therefore originally must have been part of, that empire. The RNA in these three molecules comprises about 60% of the dry weight of a ribosome. The rest comprises a few protein molecules which are simply large peptides. Individual molecules and their proteins or peptides adhere to each other and remain together in the ribosome particle. The whole ribosome particle has two parts of subunits, one larger, the other smaller. The larger subunit contains two of the three rRNA molecules and some protein. The smaller subunit is just one rRNA molecule with its proteins. One of these three rRNA molecules grasps the information RNA strand (ssBRNA in the beginning, but mRNA now) and pulls it along, one space at a time as needed, like a sort of ratchet. These movements are coordinated with the other processes happening so a move is made only at the right time. The space of each move is three nucleotides, matching the number of nucleotides in the information strand that complement and (temporarily) bind to the three on the tRNA loop. Thus, this move occurs when the ribosome has finished with one tRNA molecule. (These are sometimes called transfer RNA because they transfer an amino acid to build a peptide; a peptide that is long enough is a protein.) We should note that ssRNA generally is formed from one end to the other , nucleotide by nucleotide, and the starting end is distinguishable from the final one because the sugar (ribose) part has a different bond. (In the figure above, the green numeral 5 indicates the beginning and the 3 the end of the molecule.) Likewise the ribosome and other processes that read, examine, correct, or copy the molecule began at the 5 end, so we also follow that procedure in examining a diagram. The last three nucleotides at the end of the tRNA molecule are CCA (cytosine, cytosine, adenine) and then two atoms, oxygen (O) and hydrogen (H). When the tRNA molecule catches its amino acid, that last group of two atoms, OH (a hydroxyl radical) beinds to the hydrogen atom of the hydrozyl group of the amino acid. These

13
three atoms (OH and H) then form a new HOH/H2O molecule and the remaining end bonds then bind the rest of the amino acid to the adenine of the tRNA. Adenine triphosphate helps that process. Between the movements of the rRNA molecule (the ratchet), also from the 5 end toward the 3 end, the other two rRNA molecules release the amino acid from the tRNA molecule currently in place, attach the amino acid to the previous amino acid, if any, and notify the ratchet molecule to move forward. The ratchet also releases the tRNA molecule from which the latest amino acid has been taken so that it moves away. Thus, one by one, the amino acids are strung together into a peptide, its parts determined by the information strand through their sequences of three nucleotides each. Each tRNA molecule is released (or discharged) after completing its respective function in this project and the peptide is released when the last amino acid has been added. How does the ribosome know that the last amino acid has been added? By arriving at one of three sets of three nucleotides each, which are stop codes on the information strand. The stop codes are UAA, UAG, and UGA. None of these three codes designates any tRNA molecule. So when any of them shows up, the ribosome stop reading and peptide construction ends. If a mutation changes one of them to a set of three nucleotides that codes for a tRNA then the stop code will fail, the process will continue, and the product will either be defective or (rarely) better than the previous one. The rest of the codes are shown in the genetic code step below.

Step 37. Dependents, mergers, predators, and cannibals

Today, much writing about modern protobions emphasizes adversarial relationships between them and us, largely ignoring those that are helpful to us and those that neither harm nor help us, so we learn little about their interactions with each other. In the earliest times of their existence, they may have had little interaction because they were few, scattered, and tiny. Protobions are about one half to 2 nanometers in dimensions, roughly a billionth of a meter or yard. a. Dependents Although members of the second kingdom have been far more successful than the viroids because the reproduction starter of the latter lets them reproduce more successfully and evolve more efficiently, nevertheless, the influence of the reproduction starter of the members of Kingdom 2 can enable members of Kingdom 1 that are present to reproduce at the same time. That is, the starter for one can also start the other, if both are in the same effective environment. This fact has preserved a few viroids even to our time. In that sense, the older type has become dependent on being close to the newer type of bion to reproduce. At least one author terms this dependence parasitism, but that seems too strong. What the viroids do is more comparable to using someone elses garbage or gleaning a field of remnants too trivial to worth the owners time. b. Predators Beside this dependence, the tendency of nucleotides to link to each other also enables ssBRNAs to add to themselves (1) individual nucleotides, (2) ssRNA segments that have become separated from their original bion, and even parts taken from another ssBRNAs bion if the

14
predator bion has the ability to apply appropriate ribozymes or enzymes that would assist that attack, and the prey bion does not. This is especially true of the early and naked-strand protobions. Accidentally, this could produce a lateral transfer of a useful gene, ribozyme, sensor, etc., but the odds are against the break in the prey strand being in the right place to get such a complete, usable part. Even so, getting additional ssRNA could either help build a complementary strand for the reproduction process or add to the predators own strand additional material able to increase the likelihood of useful mutations. c. Mergers and cannibals Also, one ssBRNA could even attach itself to another, creating a single protobion larger than either had been originally. That would reduce the change of being totally destroyed, increase the possibility of useful mutations, and perhaps increase the combine differentiation of parts and functions. This would probably make it easier for a Kingdom 2 member to acquire a viroid than for any to acquire a Kingdom 2 member, because that could lead to two different starter sections in the same entity. I am not certain what would have happened in such a case in the early days. But we do know that surviving protobions today do pick up pieces of other protobions, adding them to themselves, sometimes with harmful results for humans. The mechanism used there, however, was probably not available at the time to which we refer in this volume.

Step 38. Genes.

Another process was also occurring at the same time as the development of tRNA, ribosomes, and the other steps described above from step 34 onward. Not only were the basic ssBRNAs evolving shape changes and increases in strength through development of matching or complementary nucleotide sequences within the single strand, and specialized parts developing specialized functions, but also occasional mutations or original sequences were beginning to have effects on the survival and reproduction of different lines of descent. We may, like many biologists, call various similarities species. But that term is not particularly meaningful for asexual biota and sexual reproduction did not develop until long after most of the biota of this volume were well established. That did not happen until around a billion years ago, give or take two hundred million, and we are still looking at a period three billion years before that. A better description for these older groups is lines of descent. At any rate, if a particular sequence of nucleotides, long or short and of whatever origin, prove harmful, a line of descent may become extinct. Extinctions were quite frequent in those early times, with limited abilities of a protobion to control its own fate and with a high rate of error in the replication process. When a sequence of nucleotides is useful, however, those with it will survive longer, produce more progeny, and become more numerous. These greater numbers create greater opportunities for further favorable mutations to occur. Thus, over time, a line of descent may improve its prospects and increase its ability to collect nucleotides for reproduction or enlargement, or to collect amino acids to build useful peptides. Once a particular tRNA molecule existed, any sequence of nucleotides in the ssBRNA that accidentally happened to complement those in the far loop of the tRNA could attract it and gain both the protection from mishaps by having the extract matter between the ssBRNA and the outside world as well as gaining access to a particular amino acid. Obviously, no single aspect of the several processes going on in respect to building peptides could go more than a slight distance toward that result without the others moving in

15
step. But even if the effect of each bit on survival and reproduction was tiny, say one in a hundred per generation, the generations were short and time abundant, so progress did occur. It occurred first through miniscule changes and improvements, later through repetitions of useful sections of the molecule, slowly improving coordination among processes, and gradual refinements in scope of action in the direction of specificity. Protobions were becoming able, bit by bit, to build ever more useful and accurate enzymes and structural proteins by coding parts of their main strands to control tRNA and rRNA and related processes. At that point, the part of the bion strand that codes for a tRNA that collects a particular kind of amino acid, plus the parts that code for the sections of a ribosome, can be called early genes. Such a gene would code for one product, in the early days an amino acid, but only by coding for a separate molecule from the bion strand itself. At first there was no need to code for RNA because that was part of the strand itself. The part of the bions main strand that coded for other needed tools (originally peptides) could then be called a genome, conforming to a genetic code. The author did not use either of those terms (genome and genetic code) earlier because, until the whole process of this indirect control began to form, the basic strand was not a genome or a genetic chain. Until then, it had no genes. Now that we have seen these arise, we need to examine that process from yet another angle.

Step 39. Genetic code.

As mentioned earlier, (1) the air, Ocean, and other environmental factors of our Earth caused the formation of early amino acids, at least seven of them, by or before 44 hundred millads ago (4.4 Ga); (2) these amino acids tended to join end to end to form chains, especially in the vicinity of montmorillonite clay, under the influence of this clays aluminum catalyst effect; (3) these chains tended to join at the ends into rings; and (4) those rings often had enzymatic effects similar to catalysts, some of which were helpful in the formation of nucleotides and the linking together of those nucleotides into single-file RNA strands, the first biota, the protobion Kingdom 1, the viroids. At that time there were no genes, no ribosomes, no genetic code, no specialized parts, and thus little or no control by the first bions over their own fate. At least one of them did experience a fortuitous mutation or miscopy from it viroid parent, giving it some power to initiate its own reproduction from a particular point in its strand, thereafter the beginning. Many mutations, some helpful, some deleterious, then followed, with growing population, growing useful ribozyme effects, shape changes, etc. After the first stem-loop sensor-responder formed, a series of interacting changes began to collect and influence each other, including development of three molecules which ultimately combined into ribosomes, development of tRNA molecules, ability to attract, collect, and link amino acids together, and later to influence which amino acids those would be, and in what order. A crucial part of that evolution was the origin of docking sites and genes, special sections of the ssBRNA strand devoted to parts of that control process. Of course, all these changes could only move in a useful direction by the elimination of many false starts by differential survival and by tiny intermediate steps, each gradually improving the value of the process and yielding the result of gradual acquisition of increasing influence over the fates of these protobions of Kingdom 2. Ultimately this course of coinfluencing co-evolution in all of these respects led to a fairly refined ability of the protobions of Kingdom 2 to collect the amino acids prescribed by genes formed in the B strand of the identity

16
and in the order required to make peptides (or eventually proteins) that would be enzymes assisting in needed and otherwise workable chemical processes, or useful structures. Yet one more part of this co-evolution among many factors had to be involved. That is now called the genetic code. Its foundation was the necessity and availability of the tendency of the four nucleotides then involved to match a purine (a double-ring nucleotide) to a pyrimidine (a single-ring nucleotide) and vice versa: cytosine to guanine, uracil to adenine. (I have called these four nucleotides by the names of those now in use in comparable situations. But they could have been slightly different in the beginning, as we shall see.) This matching permits and causes the template replication process, involving the formation of a new strand complementing the old strand. But it does not play any part in the relations among the nucleotides of the same strand. Now how can that be the basis of a code with only two matches? First, in a code, something must match a different thing in the real message. Suppose we write a code in which X means E in the real message. It does not follow that E in the code will mean X in the real message. It could be that S in the code will mean H in the real message. Then if the code says SX, the decoding (or real message) will be: HE. So the order or placement of the symbol makes a difference. That means in the genetic code, A C G U in the code or main information strand will match U G C A on the decoder or message, the complementary nucleotide sequence. So, not two, but all four nucleotides make the foundation for the code. Will that be enough? It probably helped in the beginning. The system arose bit by bit, so perhaps an adenine on the main strand might mean simply, bring me an amino acid, if an early precursor to tRNA could do that. At the next stage, as previously stated, it might help to say, bring me a polar amino acid (or bring me a nonpolar amino acid). Then a second nucleotide could become a standard part of the code, distinguishing between these two types, say, either cytosine or guanine. Then the two-part code AC might mean, bring me a polar amino acid, while AG might mean, bring me a nonpolar amino acid. There may not have been many amino acids then, or not many were in demand near a protobion. Some theorists suggest there were between six and ten. If one of these was the only one in short supply, and others were present all the time anyway, then maybe one nucleotide alone could specify that one amino acid, say, uracil. Two related problems, though, arise with that simply code. Mutations occur and the information strand is full of all four of these nucleotides. One symbol alone would be too undependable and would make a weak link with the pre-tRNA. So that stage would likely soon be replaced by a two-symbol (or two nucleotide) code. The code symbol on the bions main strand, the information strand, would have to be adjusted to match at least two of the nucleotides in the part of the tRNA molecule that would dock on the pertinent part of this gene corresponding to the position of the needed amino acid in the sequence of the desired peptide. So, if the pre-tRNA has on its docking part (the middle of the farthest loop) has, say, CC, the budding gene section will, in time, adapt to that with the code CG. (These particular choices were not necessarily the ones that really were used. These are just theoretical examples to express the workings of such a system.) What is clear is that all the structures, molecules, codes, etc., could only evolve from a simple beginning by small steps that would increasingly adapt each of them to all, and at each intermediate step would improve the survival or reproduction rate of the bion. As the system grew, its specificity increased, the number of amino acids ushered about and used enlarged, and the code merely amounted to designating the tRNA molecule that would bring back the next amino acid that was needed.

17
One may liken the situation to a row of simple-minded supervisors, each of whom knows the name of only one member of a crowd of fetchers standing around in front of the row. Each supervisor in this situation can only speak when a manager, walking behind the row, touches him on the back. The manager starts at the beginning of the row and touches each supervisor in turn as he goes. The supervisors, in turn, each speak the single name known, George, or May, or Aloishes, etc. Each fetcher then hands a wrapped package over to the supervisor who called him. In this analogy, the manager is the ribosome, setting the pace and maintaining the order in which the fetchers do their job. The supervisors are the nucleotide sequences that code for the required amino acid. The name that each of these supervisors pronounces identifies the fetcher who is to act next. The fetcher is a tRNA molecule who brings a package, but only one kind of package. And the package brought is the specific amino acid that the particular tRNA molecule can bring. So we call it a code only because that is a short word that describes what it means to us. To the bion it is not a matter of meaning, though. The fetcher (the tRNA molecule) brings one kind of amino acid, the gene or docking station nucleotide sequence that matches the fetcher. It is working system in reality, not a communication system. But we can think of it as information being communicated. The system evolved into being and, over the millads of time, it is still evolving. Most bions today use and recognize (or respond to) the same basic code. But some differences do occur. In modern tRNA molecules, uracil is delivered for their construction, like the other nucleotides previously mentioned. But in numerous cases the uracil is then modified slightly. The changes do not alter where or how the molecule fits, but technically changes its identity. In one case the uracil changes to thymine, which otherwise does not appear in protobions or their products. Inosine is another unusual nucleotide that is sometimes a result of changes during construction of tRNA molecules. From this I would infer that these tRNA molecules once had a slightly different component that is not now available. So building them requires modifying a present-day product to what was used in the past. Odd forms like that are rare in other biota or biological products but not unheard of. Some ancient life forms, like the archaebacterium41 that has become an organelle called mitochondrion in each of our cells has an odd form of this type.
41

In the past, a group of single-celled microorganisms thought to be a subtype of bacteria was classed along with bacteria as prokaryotes and named archaebacteria. This subtype is now classed and named differently. The subtype is named Archaea, which form an independent domain (a grouping that this author terms an empire). These microorganisms are mostly similar to bacteria is size and shape. But their genes and metabolism are more closely related to those of eukaryotes. In other characteristics, these organisms are unique, allowing them to use a wider range of energy sources. Some rely on sunlight as this energy source, others fix carbon, other use organic compounds such as sugars or even ammonia, while still others depend on metal ions or hydrogen gas. See Wikipedia article on Archaea for additional details. The newer classification is based on the work of C.R. Woese, O. Kandler, and M.L. Wheelis. 1990. Towards a natural system of organisms: proposal for the domains Archaea, Bacteria, and Eucarya, in Proceedings of the National Academy of Sciences U.S.A. 87 (12): 4576-4579.

It is unnecessary to go into details about the evolution of the genetic code. Two significant aspects should be noted, however. One is that the genetic code seems to have had a period when one message unit, a codon, comprised of two nucleotides, but later changed to three as it has today. So, three nucleotides constitute a codon which specifies a particular tRNA and thus indirectly a particular amino acid. There are, as previously stated, 20 amino acids in

18
common biological use. (A few additional amino acids are used by a few biota, but 20 are common.) The result is 64 possible codons. Each tRNA molecule is limited; it can collect only one type of amino acid, but the amino sometimes more than one tRNA can collect a particular type of amino acid, because a different codon is used that only a different tRNA can use. Three codons do not represent any amino acid or tRNA molecule. The ribosome reads each of those three codons as the end of the message, called a stop codon, and discontinues its action at that time. The second significant inference reported from comparison of various aspects of the genetic code is the implication that, because of the proportions of various ingredients, the Ocean was more acidic four billion years ago than it is today. Perhaps that was because the concentration of phosphoric acid was greater then, though other causes could be imaginable. Phosphoric acid seems most likely to me. If there was more phosphoric acid before biota arose and in the early few hundred millads when only protobions existed, its role in the origin of earthly biology may have been even more important than suggested above.

Step 40. Combined and cumulative effects: growth, collection, and protection.
Recapping, chapter D briefly outlined the formation of the Sun, Earth, Moon, and the early phases of geology. Chapters E and F recounted a rough summary of pre-biological chemical changes that led toward the formation of the first bion. (1) These chemicals included four of the six most crucial elements present in the early air, carbon, hydrogen, nitrogen, and oxygen. Some of their important compounds were described as well, such as ammonia (NH3), carbon monoxide and carbon dioxide (CO and CO2), cyanide (HCN), methane (CH4) and other hydrocarbons, water (H2O), etc. These underwent (2) various chemical changes that resulted in amino acids, aldehydes, sugars (ultimately including ribose), and perhaps two of the four early heterocyclic organic bases, through a combination of atoms from the air and the water, mainly of the Ocean, with its heavy doses of ions of many types (including sodium, chlorine, iron, and many others), forming salts and other compounds. (3) Some of these were carried by Ocean currents and other influences to the geothermal vents and plumes on the Ocean floor, where there were hot gases, water, and various chemicals, including phosphates, more carbon dioxide, sulphates and sulfides, iron and nickel, etc. (4) Many of the molecules of the Ocean floor were catalysts and sources of high chemical energy, such as the aluminum clay that assisted early amino acids in forming enzymatic peptide rings. The latter, in turn, likely influenced the formation of some of the steps in the formation of the remaining organic bases of the nucleotides. The phosphates and iron and nickel compounds also likely played similar parts and, of course, the phosphates constituted a necessary part of the nucleotides themselves. These and other atoms and molecules likely also played a part in assisting the nucleotides, once formed, as did some nucleotide action itself, (5) to assemble those nucleotides into chains or strands that constituted RNA and the first bions, the viroids. (6) At least one of these drastically limited viroids evolved the ability to start its own reproduction or replication by matching up with collections of other nucleotides that would complement its own, in the same order, and influenced these matching nucleotides to link themselves into a new strand. That was a crucial great leap forward, and established the second kingdom of biota, the ssBRNA. That second kingdom rapidly grew, diversified, and added new capabilities, including (7) strengthening and shaping themselves with double-stranded stems resulting from complementary sections (each the reverse of the other) of the main strand attracting each

19
other; (8) evolving parts of themselves into ribozymes or sections that could influence the behavior of the protobions themselves or other molecules; (9) converted their stem-loop structures into sensor-reactors to certain molecules and conditions; (10) gradually evolved a whole collection of parts and abilities (fetchers of amino acids, a three-part ratchet, a detacher and reattacher, etc., thus making use of these amino acids, docking stations for the fetchers, genes, and a genetic code), each affecting the others, that ended in enabling bions, for the first time, to create their own peptides, both as enzymes and as structures. There were, of course, other improvements in the ability of the ssBRNA to survive, multiply, and progress further. The next chapter will touch on some of that progress. All of this tends to show that the first bions did arise from organic, but not biological, chemical changes in a particular set of environments, that they have evolved since then, fairly rapidly in that early half-billion year period, and have survived to our own time, despite their limitations. They could not yet fully control their own fates but they took several steps in that direction. Once they could make peptides or proteins, they could produce them repeatedly in larger numbers, they had the necessary material to work more productively and faster than the single-stranded RNA could by itself as a set of ribozymes, although they retained that power too. Still, they could not control their energy sources at first, could not yet plan or even eat, although they could add nucleotides to the main strand. Their replication was not exact and many occurred. That meant that many were defective and could not survive or, perhaps, even function. But the inexact copying also led to very rapid evolution in response to competitive pressures. As the ssBRNA grew longer, they could develop more capacities, but they also became more vulnerable to disruptions. Then a new ability developed. Once these protobions could cause the formation of specific peptides, not only could enzyme helpers be built, but they could also make peptides that formed structures. One of those structures that became common among protobions was a protein container called a capsule.42 A certain type of peptide or protein molecule was formed that tends to join others in specific orientations. These peptides would then automatically join together into a single, solid box or container, rather like a geodesic dome. The most common form of these today is an icosahedron, with 20 flat, identical faces. Of course, other shapes exist as well.
42

In biology, a capsule is the shell of a bacteria made up of polysaccharides, long molecules of monosaccharide units joined together (see Wikipedia article on bacterial capsule and polysaccharide). The term capsid is used for the shell of a virus and is made of protein units called protomers. At this point in the authors description, only the capsid has evolved; the polysaccharide capsule will come later. However, this author often uses terms somewhat differently from accepted usage, especially when he feels this usage is misleading or confusing. So I have left his wording unchanged.

Presumably an attraction arose between these capsules and the RNA strand so that either the capsule forms around a strand, enclosing and protecting it, or the strand enters the capsule when formed, even though it appears impenetrable. In addition, some protobions after this invention could and did extend their strands farther or even break them up into several parts, because all the parts could be kept together inside the capsule. At least one such bion even makes a series of capsules, one inside the other, like Russian nested dolls, with a different RNA strand in each capsule! In later times, capsules evolved with many different shapes and forms and other progress occurred. We deal with those later. Before that, we must look at new kingdoms.

20 Chapter H. Steps 41-50 The First Empire Grows.

Step 41. Revisiting bion structure, functions, and genetic system.
We have seen that the first bion was simply a naked strand of ribonucleic acid nucleotides, with great potential but virtually no developed capabilities except the passive but crucial one of possibly being the template against which a collection of free nucleotides could line u and assemble into a complementary strand as a sort of offspring. At least one of the members later (but probably very soon) added to this start the ability to initiate that matching process, at a particular point, and then to help the unification of the new strand, and still later developed stem-loop sensor-responder parts of the strand, enzymatic parts of the strand, other special structures, and a genetic system with multiple interacting parts. The simplest of those first bions, to the extent that they survived to our time, were small compared to later bions, but probably had at least a few hundred nucleotides, as they still do (generally fewer than 400). Once the second kingdom of the protobions formed, that number gradually grew (not in every lineage, but in many), as the bion strand became not merely one of countless kinds of molecules in the Ocean, but one which could increase its likelihood of survival and reproduction by devoting part of the chain to replication initiator, stem-loop structural stiffeners, sensor-responders, ribozyme sections, genes, and other special sections. The protobions themselves, each made of a single strand of naked RNA, not only had these growing capabilities but also access to a considerable range of allies in the environment. Those allies included the still independent nucleotides, which could (1) serve as raw material to build new protobion complements of themselves; (2) become additional parts of an existing protobion; and (3) help provide energy to power various biological processes needed by the protobions, especially mergers and the building of new structures. Other such allies included various cracks and crevices in rocks, pores in the soil, and spaces around sand grains on the Ocean floor, in all of which mishaps might be reduced to some extent. Inorganic catalysts also helped, including metal ions in solution and metal atoms in compounds with surfaces, such as an the Ocean floor. Of assistance as well was the occasional and fortuitous, favorable, enzymatic action by ring peptides of modest size, formed by pre-biological processes. In addition, high temperatures and copious minerals provided by the geothermal vents and plumes on the Ocean floor helped. These minerals included sulfur, copper, phosphorus, etc. At some point, for example, sulfur assisted bions in cross-linking certain proteins to guide and strengthen their folding property (necessary to function) and otherwise provided the chemical characteristics of oxygen where oxygen molecules themselves would have been too chemically enthusiastic. We have already met the various parts of the genetic system that gradually developed by little changes in various different respects, each responding to some degree to the environment created by the prior changes. It may be fitting to refine some of that information as a preparation for the next step. The earliest stages in the development of tRNA remain somewhat speculative, but it is reasonable to suppose that after this structure had gone beyond the usual size and nature of sensor-responder within the main chain the rest of the chain annealed itself together, omitting the tRNA as it was then, however and for whatever reason that happened, two or three nucleotides on the loop of this new molecule tended sometimes to find matching (or complementary) nucleotides on the main strand and pair up there. Even if no other function

21
was present, such an arrangement could provide some protection to the bion strand simply by being in the way of an approaching molecule that would otherwise prove harmful. The tRNA molecule could be expendable from the viewpoint of the bion strand, providing an advantage in both making more such extra molecules, to stud the main strand with such a protective covering. After all, a tRNA molecule has fewer than 100 nucleotides, while the smallest protobions had hundreds, the more advanced protobions of the second kingdom generally had thousands, and some had tens of thousands. That arrangement would also provide strong survival pressure toward matching two or three nucleotides on the outer loop of the tRNA molecules with three on the main strand (as docking sites) to make the system work. That much must reasonably be inferred to have happened during and out of the protobions of Kingdom 2, for the following reasons: (1) both the main bion strand and the tRNA satellite product consisted of a single, naked strand. (2) This was a necessary prerequisite to gaining control of the nature of the peptides formed thereafter. (3) Some single-stranded bions later made their own protein capsids (enclosures) to protect them, but could not have done so before they had gained control of the construction of peptides of the necessary types. The most common of these had only a few proteins, which automatically formed plates of protein around their RNA single strands. The plates were all the same size and shape and automatically assembled into the enclosing capsids. In view of the most likely sequence of events, requiring the foregoing steps to occur before the ribosome could form very effectively, the ribosome probably formed a little later. In the process, perhaps the steps at the ends of each tRNA strand may have gone from (a) development of adaptation to stopping acids or ammonia to (b) collecting amino acids to (c) collecting categories of amino acids to (d) collecting a few especially needed specific amino acids to (e) collecting all specific amino acids needed. As it is, bions still do not use all the amino acids that exist. One authority on genetic-code evolution concludes that, before the final, unmatched sequence of CCAOH (an RNA molecule ending with two cytosine molecules, one of adenine, plus an oxygen atom and one of hydrogen) was added to the main stem of tRNA, that stem was a helix (a coil of twisted RNA) containing nucleotides that attracted suitable amino acids.43 Later, when the system was efficient enough to create specific peptides (protein enzymes), the CCAOH could be added to attach all of them, and the proteins did that part of the job. (See figure in step 35 above.)
43

It is not clear to me who this authority is. For a historical summary of the view that an RNA-based biology preceded that based on DNA, including a proposal that molecular systems capable of replication may have preceded RNA, see Cech, Thomas R. 2012. The RNA Worlds in Context in Cold Spring Harbor Perspectives in Biology 4: 1-5. doi:10.1101/cshperspect.a006742. In the latter article, Cech cites experiments by Schrum et al (2010) in achieving replication of simple nucleic acid -like polymers within lipid envelopes, thereby constituting protocells that can grow and divide. Hence, see Schrum, J.P., T.F. Zhu, and J.W. Szostak. 2010. The origins of cellular life in RNA Worlds (eds. J.F. Atkins, R.F. Gesteland, T.R. Cech). Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press.

The three molecules that ultimately became parts of the ribosome, probably already in existence and beginning to adapt to their roles, gradually improved and ultimately combined to do the job. Thus, these special molecules (dozens of tRNAs, three rRNAs, and in time various peptides and proteins), as well as the genes and the genetic code, began to emerge. Today, two of the three rRNA molecules join to form one part of the ribosome and the third forms the other part. When some tRNA molecules begin to assemble on the strand, which provides them the

22
information of the specific docking sections in order (originally the genes, but today a separate strand like the original but with sites only for one peptide or polypeptide protein molecule to be made), the two parts of the ribosome assembly, do their processing steps, and then the two parts separate until needed again.

Small subunit ribosomal RNA, 5 domain (from Wikimedia Commons, taken from the Rfam database). This example is RF00177. The whole RNA molecule has three additional domains, a central domain, a 3 major domain, and a 3 minor domain.

The ribosome functions and process are more complicated than is useful to describe here in further detail. Historically, though, it seems useful to touch on the structure of the three molecules that compose the ribosome. (Today they are accompanied by a number of protein molecules that form firm, compact objects within the modern ribosome; but these could not have been present at the beginning.) The enzymatic functions of the ribosome, influencing the chemical reactions taking place, are performed by the rRNA molecules as ribozymes, so the role of protein molecules here seems not to be enzymatic but structural, stabilizing the complex rRNA molecules of the ribosome.

23
First, like the ssBRNA strands and tRNA, the rRNA molecules are each a single strand. To perform their complex task, however, they need to hold a specific and fairly complex structure. The proteins seem to help in that but, by themselves, they are not enough. The rRNA molecules therefore double back on themselves, held that way by complementary nucleotides, for most of their length, much more so than the tRNAs. See the figure above for a portion of the smallest rRNA molecule. Second, let us remember that the ribosome consists, in its simplest form, of three rRNA molecules, two in a larger portion (or subunit) and one in the other (also with the proteins). The two portions assemble together only when needed (under the influence of a chemical signal), perform their function, amino acid by amino acid, until the peptide (specified by the coded docking stations, in order) is formed, and then separate until the next call for their services. Let us also note some features of the genetic code. As discussed before, the code (at its simplest) now consists of the four nucleotides adenine, cytosine, guanine, and uracil, with the first and last complementing each other, and the two middle ones complementing each other (usually abbreviated as A, C, G, and U). (Biochemists early in the 20th century thought these four were too few to form a genetic code but, in mid-century, a new generation proved this sufficient.) Each minimum message, a codon, has three of these nucleotides lined up side by side. It happens that a code of only four elements (which we may think of as four letters), which must be taken in codons of three elements (or letters) each, provides 64 possible codons. Three of these codons, it turns out, do not code for any tRNA and thus not for any amino acid. The ribosome treats each of them as a stop code. When one shows up, the ribosome stops its activity, releases the newly made peptide or protein, and breaks into its two portions. A mutation that substitutes ones of these three codons for a coding codon makes the resulting peptide or protein defective, which may prove immediately fatal or else slowly harmful to the bion with this defect.

Three-dimensional depiction of a ribsome in two views, showing rRNA in blue (small subunit) and red (large subunit). Lighter shades represent proteins (from Wikimedia Commons, article Ribosomal RNA ; originally from Rfam at rfam.sanger.ac.uk/family/RF00177).

Individual bions, or species of bions, typically do not use all 64 possible codons, but some amino acids are located by more than one kind of tRNA molecule. The three nucleotides on the main loop of a tRNA molecule will find and dock at the docking site (or binding sites), or three nucleosides complementary to its docking three. For a few examples, suppose the docking code on the tRNA is AAC. Then it would dock at GUU on the information strand (which was usually the B strand in early protobions). Note that the two strands run in opposite directions, each from its own 5 to its 3 end, in order to match up, just as would happen in replication. The newer ability arises, as normally, out of the older one. Consequently, the two codons must also run in the opposite direction. In our example, the docking would be as follows:

24
5 AAC 3 (code for tRNA) 3 UUG 5 (code for main information strand RNA) In the main information strand RNA, its codon reads GUU (the opposite of what we read here) from its own view. That is because of the direction of reading, shown as left to right for the tRNA, right to left for the main information strand RNA. It often happens that two or more kinds of tRNA (in one case, six!) fetch and deliver the same kind of amino acid. The information strand often also has two or more codes to accommodate these transfer molecules. But in some cases two (normally not more) different kinds of tRNA molecules can recognize the same set of three nucleotides at a docking site. In such cases, two elements of the information strand do complement the opposite two on the transfer molecule, which is said to display wobble in docking either at a fully complementing triple44 or at a certain one which does not complement the third position. This odd feature still gives the same amino acid as specified, so it does no harm, and thus surely arose in later evolution as a helpful feature, reducing delay and excess production of tRNA molecules.
44

The author may mean third rather than triple here.

(As a minor note, the reader may also find it of passing interest here to know that alanine was the currently used amino acid most common in the original Miller-Urey experiments and is found in carbonaceous chondrites, a kind of meteorite that frequently falls on our Earth from interplanetary space and possibly, on occasion, from farther away.)

Step 42. Third kingdom of the protobion empire.

a. Preliminary remarks. Formation of the first protobion established the existence of the individual, and the first offspring established the replication by template use of ribonucleic acid, producing a complementary strand (a matching one, with G for C and vice versa, A for U and vice versa). That group of protobions deserves to be regarded as a separate kingdom because they are unlike the members of any other biological kingdom, then or now. They also clearly do not belong to either of todays groupings of similar kingdoms, which I have called empires and some others realms.45 The protobiota were in their original time the only biology on Earth, of course, and even now the only ones that are not cells. Unlike cells, they are not surrounded by a protective membrane, a cell wall, or any other fencing that separates them from the open Ocean or the air. Yet they are distinct and identifiable. One authority reports that they remain today about ten times more numerous than the next most numerous forms of bion, both in a given amount of Ocean water or in the same amount of soil.46 (Of course, although much larger on average (certainly in the largest forms) than their early ancestors, they remain much smaller individually than other bions and therefore their total mass is much less.)
45

The more common term for what this author calls an empire appears to be domain. For example, in the Wikipedia article on Virus (en.wikipedia.org/wiki/Virus), there is the statement: Viruses are now recognized as ancient and as having origins that pre-date the divergence of life into the three domains (citing Mahy, W.J. and M.H.V. Van Regenmortel, eds. 2009. Desk Encyclopedia of General Virology. Oxford: Academic Press, p. 28). The protobiota of which the author has spoken thus far are termed

25
viroids. In the Wikipedia article on Virus (see previous citation), there is this definition: Viroids are molecules of RNA that are not classified as viruses because they lack a protein coat.They do not code for proteins but interact with the host cell and use the host machinery for their replication (citing Tsagris, E.M., A.E. de Alba, M. Gozmanova, and K. Kalantidis. 2008. Viroids, in Cellular Microbiology 10 (11): 2168. Doi:10.1111/j.1462-5822.2008.01231.x. PMID 18764915). Virion, in contrast, is defined in the same article as a single, stable infective viral particle that is released from the cell and is fully capable of infecting other cells of the same type ( 46 I am uncertain of the identity of this authority. Proof of the viroids physical nature was demonstrated by T.O. Diener (1972. Potato spindle tuber viroid. VIII. Correlation of infectivity with a UV -absorbing component and thermal denaturation properties of the RNA, in Virology 50: 606-609). C. Hernndez and R. Flores (1992. Plus and minus RNAs of peach latent mosaic viroid self-cleave in vitro via hammerhead structures, in Proceedings of the National Academy of Sciences U.S.A. 89: 3711-3715) divided the viroids into two families with of the 30 or so known species belonging to the family Pospiviroidae, four others belonging to the family Ausunviroidae (for a brief summary of the field with references, see also Dars, J.A., F.E. Santiago, and R. Flores. 2006. Viroids: an Ariadnes thread into the RNA labyrinth, in EMBO reports 7 (6): 594-598). These numbers fewer than 50 species do not substantiate the authors description of the ubiquity of viroids (his protobions). It may be that the author means to speak of viruses. The Wikipedia article Virus (citation in footnote above) states There are millions of different types of viruses, although only about 5,000 of them have been described in detail (citing Casjens, S. 2010. In B.W.J. Mahy and M.H.V. Van Regenmortel, eds. Desk Encyclopedia of General Virology. Boston: Academic Press, p. 167; see also Dimmock, N.J., A.J. Easton, K. Leppard. 2007. Introduction to Modern Virology (sixth ed.). Hoboken, NJ: Blackwell Publishing). The same article further states: A teaspoon of seawater contains about one million viruses (citing Shors, T. 2008. Understanding Viruses. Jones & Bartlett Publishers. p.4)

So far, I have divided the early protobion empire into two kingdoms. The first was the virtually helpless, naked, single-stranded BRNA molecule long enough to leave functioning descendants, which I have called viroids, because that term is used today for some of their descendants. They had no genes, made no products, and could not reproduce very effectively without help from a bion of a more advanced kingdom. But at least two other groups of essentially the same kind of minimal bion have received different names from biologists. One such term is co-virus (defined in the Oxford Dictionary of Biochemistry as any virus that exists as two or more separate particles, all of which must be present together in the host organism for the complete replication cycle of the virus to occur). The other is such a narrow category that we will not bother with the term here. The same basic description, though, fits all three.47 So here we shall henceforth use a new title for members of Kingdom 1: bioids (pronounced by-oids). Kingdom 2 of the protobion empire consists of the remaining single-stranded BRNA bions (aside from the bioids). This time has come to introduce the third kingdom in this same empire, the double-stranded BRNA bions.
47

G. Bruening (1977. Plant Covirus Systems: Two-Component Systems in Comprehensive Virology II, H. Fraenkel-Conrat et al., eds. New York: Plenum Press, pp. 54-55) notes that a two-component virus may be made of two independent viruses, with two chromosomes that differ from each other, but which together may have properties that neither does alone. These essentially independent viruses that happen to work together may be termed hybrid viruses or pseudo-recombinant viruses. There are three groups of two-component virus systems: tobraviruses, comoviruses, and nepoviruses. One or more of these terms may be the present authors narrow category. Note that a previous chapter in the same volume describes three-component viruses (Van Vloten-Doting, L. and E.M.J. Jaspars. 1977. Plant Covirus Systems: Three-Component Systems in Comprehensive Virology II, H. Fraenkel-Conrat et al., eds. New York: Plenum Press, pp. 1-53).

26
It is easy to imagine three or more possibilities for deviations from the standard Kingdom 2 or ssBRNA format. All three do, in fact, exist. (1) One part of the single strand may fold back against another part because the two complement each other (though the complementing parts would have their complementing nucleotides arranged in opposite orders). This happens in stem-loop structures, of whatever function. It is true of the four-system parts of a tRNA molecule and of so much of the rRNA molecules that, at first glance, they appear to have two separate strands. Yet in all these cases, despite the complementing binding, only one strand is present, as is obvious if one looks at the two ends of the single strand. This is still a single-stranded BRNA. (2) For whatever reason, individual free nucleotides in the vicinity, or small sections already assembled together, may find that they match or complement one or more sections of the main single strand and therefore bind to it. These sections could thus become double stranded, but would not last long in the competitive conditions of the Ocean after bions became numerous, unless the double-stranded sections somehow increased the likelihood of survival reproduction. An alternate occurrence that might not be distinguishable from this in retrospect would be a folding back of a single strand on itself, followed by a break at the end. Various examples of different degrees of an end result have been found and either cause for them may have applied. If the divergence from a normal ssBRNA is regarded as sufficient, these are called transitional forms. They are treated as preliminary forms of a third kingdom of the protobion empire, the dsBRNA, double-stranded Bion RNA. (Although these protobions all have genes and hence genomic parts of their RNA strands, other parts remain reserved for other purposes, such as initiating replication or reproduction, identifying its starting point, ribozyme functions such as influencing the collection of nucleotides for reproduction, linking them to each other, and releasing them for the parent strands, etc., although many of these functions gradually were replaced with genes controlling the creation of peptide enzymes.) (3) Most importantly, all bions that successfully reproduce do so by matching new, complementary nucleotides, running in a reverse direction to that of the old strand(s), against their existing strand(s) which serve as the template. It is easy to see that some defect in the process could prevent the newly created and added strand from leaving the old one in an Ocean environment. The result would be the creation of a doublestranded molecule from what had been a single-stranded one. This, too, most likely happened and probably was the most important source of double-stranded RNA molecules and bions. These are called, as noted, dsBRNA. b. Advantages and disadvantages Clearly protobion Kingdom 2, as we have seen, was a great advance over Kingdom 1, and led to far more potential and actual accomplishments and far more bions. Kingdom 3, the dsBRNA, is not necessarily so clearly an advance. A double-stranded bion is clearly more stable and robust than a single strand because each helps to hold the other and all of the nucleotides together. These additional bonds make it very strong. This is an advantage. On the other hand, double-stranded RNA normally does not easily separate back into two single strands (that is what makes it strong). But that separation is necessary to permit copying or replication (template-matching reproduction), especially in the Ocean. Early protobions were normally in the Ocean. It happens, though, that one example of a double-

27
stranded RNA protobion had already evolved the ability to create a protein capsid in which its RNA could safely reside. Evidently that capsid was small and sufficiently water-resistant to keep its RNA fairly dry inside. That relative dryness kept water molecules away from the hydrogen atoms in the RNA strands. When this bion first produced a double-stranded form, it was already in that capsid, where the lack of water takes away the already limited strength of the hydrogen bonds between the two strands, so that separation could occur there and the separate partners could then join or build new complementary strands when released from the capsid. This kind of situation is called pre-adaptation, meaning that a change in circumstances reveals (or creates) a new advantage to a characteristic already present (and serving some different function previously). For this reason, this bion survived to our own time, without drastic further improvement, but its ancestor could have been ours as well. There is no strong evidence that it was or that it was not. Other dsBRNA protobions also exist and probably more existed at one time, all able to survive because of particular adaptations to overcome the disadvantage of the limitations on separating the two strands for reproduction. Although an advance in strength of the individual, the difficulty of reproduction seems likely to have made this bion an evolutionary dead end.

Step 43. Other capsids.

Besides the common, simple, 20-faced, icosahedral capsids of some protobions, previously mentioned, other, more complicate capsids have come into existence, usually accommodating larger and more complicated protobions. All of the reported capsids are made up of protein molecules that self-assemble into their ultimate combined structure because of their chemical properties. These additional capsid shapes include spheres, cigar shapes, and a few others with discrete, flat surfaces. The largest tend to have more than one layer, rounder shapes (mainly because multiple faces make the whole shape appear more rounded as the faces become too small to distinguish), more components, and sometimes extra molecules contained inside (see next step), besides the nucleic acid strand(s). As mentioned previously, RNA strands able to reproduce at all normally contain a few hundred nucleotides, fewer than 400 in Kingdom 1. In Kingdom 2, these strands contain from several hundred to thousands of nucleotides (in one case, for example, 7700 nucleotides). Those in Kingdom 3, just described above, contain tens of thousands (one, for example, has about 23,000).48 As the numbers of nucleotides and length of RNA strand increases, more sophisticated means develop for protecting the strands, keeping them from tangling (or separating if the strand breaks), or interfering with their own functions. The RNA strands do tend to form a helix or spiral where they double up, whether as parts of a single strand or as separate strands in a double-stranded form, and that helix helps to maintain both order and strength. As the strand grows, normally the genome part becomes more significant and the ribozyme part less so; likewise, the capsid must become bigger.
48

The smallest viral genome is that of ssBDNA circoviruses (single-stranded bions with DNA rather than RNA). Their nucleic acids code for two proteins and the genome includes 2 kilobases. The largest viral genome occurs in the mimiviruses which have genomes of over 1.2 million megabases and which code for over a thousand proteins (from Wikipedia article on Virus, citing Rybicki, E.P. 1990. The classification of organisms at the edge of life, or problems with virus systematics, in South African Journal of Science 86: 182-186). RNA viruses may segment their genomes, forming two-component and three-component systems, apparently reducing the rate of fatal replication errors and creating hybrid forms.

28

Step 44. Enzyme in the pocket.

Some protobions that have capsids have been found to package already made enzymes in their capsids along with their nucleic acid. This is a clear step forward and important. Although protobions still in existence do not normally carry on active processes involving their nucleic acid within the capsid, containing some enzyme molecule(s) makes these protobions immediately ready to use the enzyme(s) as soon as the nucleic acid leaves the capsid or the capsid breaks up, which is necessary before reproduction of such a bion can occur. Containing these enzymes also involves a supply of something needed other than the nucleic acid, something not typical of most surviving protobions. Hence, such a store is a step toward more advanced biota, even though only a tiny step.49 I have seen no reports of multiple, different enzymes kept within the same capsid, but not all of the largest protobions existing today have had all their genes and other details reported. I would not be surprised if some have more than one type of enzymes, perhaps a number of different types, and in pre-cell days this must have been more true than now, because it would have been more valuable for the early bions to have these enzymes.
49

Various types of enzymes that aid replication have been found inside various types of viruses, including: polymerases which synthesize RNA and DNA (see Wikipedia article RNA_polymerase citing Ahlquist, P. 2002. RNA-Dependent RNA Polymerases, Viruses, and RNA Silencing, in Science 296: 1270-1273), replicases which copy RNA (Wikipedia article RNA polymerase citing, among others, Zanotto P.M., M.J. Gibbs, E.A. Gould, E.C. Holmes. 1996. "A reevaluation of the higher taxonomy of viruses based on RNA polymerases," in Journal of Virology 70 (9): 608396. PMC 190630. PMID 8709232), and reverse transcriptases which synthesize DNA from RNA (the reverse of the usual transcription, hence the name). On the latter, see Wikipedia article Reverse transcriptase (citing the article Retrovirus at www.biomedicine.org/biology-definition/Retrovirus/).

Step 45. Protein stabilizers or spindles.

A few protobions have even been reported to have special protein within their capsids that strengthens the stability of their nucleotides. Most reported protobions do not, and in fact even the next more complex bions (compared to protobions) include some species which do not display such a characteristic. But our human cells do have proteins preforming similar duties and so does one kingdom in the second empire (the latter containing the first cellular bions).50 The class of proteins preforming this stabilizing function in us human is called the histones and that is true of the one kingdom in the second empire that generally has such proteins. The human histones, in addition to stabilization, have also been reported to perform some regulatory functions with the aid of (i.e., in response to signals from) small RNA molecules. So it is possible that something similar is true of one or more protobions. It is also possible, although unlikely, that a surviving protobion might have a protein in use in this way which is an ancestor (or at a descendant of an ancestor) of our histone. That, however, has not been shown. It would be fascinating to discover, if true.
50

A histone is an alkaline protein molecule that acts as a spool around which DNA is wrapped as the thread (Wikipedia article Histone). Histones now occur in the nuclei of all eukaryotic cells (i.e., cells with nuclei) except for those in dinoflagellates (a type of algae), as well as in some Archaea, specially the Euryarchaea (citing Youngson, R.M. 2006. Collins Dictionary of Human Biology. Glasgow: HarperCollins;

29
also see Cox, M., D.R. Nelson, and A.L. Lehninger. 2005. Lehninger Principles of Biochemistry. San Francisco: W.H. Freeman). These Euryarchaea may be the protobions of which the author speaks.

Step 46. Kingdom 4 of the Protobion Empire: the switch starts.

Once protobions existed as single-stranded RNA molecules able to initiate and begin to control (by designating the starting point) their own reproduction (actually, the building of a complementary strand), biological change and further progress came quite rapidly, resulting in many small and several major transitions in quick succession (within a few million years, probably, although precise times are not yet proven. Yet it cannot have taken very long unless the earlier and chancier changes were much faster than suggested here, because we know the biggest changes that are specifically certain must all have been past before 3.9 billion years ago, or at the very most perhaps before 4 billion years ago.51
51

I am uncertain of the source of this certainty that the change from RNA to DNA preceded 3.9 Ga. The Wikipedia article DNA, in discussing evolution of this molecule, states that the it is unclear how long DNA has existed because this molecule degrades over time, surviving less than one million years (citing Lindahl T (1993). "Instability and decay of the primary structure of DNA". Nature 362 (6422): 70915. doi:10.1038/362709a0. PMID 8469282).

Within that brief time, another kingdom arose, Kingdom 4, which we may call the nucleic acid switch or transition. This kingdom again, like Kingdom 3, has a double-stranded BRNA basic identity, containing its growing set of genes, its start and end sections, any surviving ribozyme parts, if any, and perhaps a few other, smaller sections. It also has a capsid to contain, protect, and preserve its two strands, running, as usual, in opposite directions. The whole structure of the body of this protobion also includes a number of other structures associated with the capsid, some of these inside the capsid, some of these outside and around the capsid. These structures differ slightly among members of this kingdom, as shown in an accompanying figure. What makes this set of protobions different from Kingdom 3? The crucial and defining difference is that, in reproducing descendants, these transitional bions introduce an important and dramatic, new step in the process. Instead of just having its two RNA strands separate, with each part acting as a template to form new RNA copies of the other, it substitutes a new kind of nucleic acid as the material in which to form new copies: deoxyribonucleic acid (DNA). As the deoxy- indicates, the only chemical difference between the new DNA and the old RNA is that the five-atom sugar ring that holds the phosphate ion to the organic base in DNA has one less oxygen atom attached to the outside of the ring (not part of the ring) than in RNA. That one less oxygen atom is the one that in RNA is bonded to the carbon atom of the ring next to the carbon that binds to the organic base, as shown below (only the sugar part is shown; the phosphate and organic bases are not shown):

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RNA chemical structure (from Wikimedia Commons).

DNA chemical structure (from Wikimedia Commons).

What would be the reason for such a change? In the first such event, of course, as in all of evolutionary biology, the loss of one oxygen atom was, from the point of view of the bion, purely an accident. For the change to be retained, it would at least need to have no cost exceeding any gain; in this case, adding another step to the process was a small cost but still less efficient seeming, so there must have been some advantage. RNA became the first truly independent biological molecule because it could form from the resources and circumstances available, it could (1) act as a ribozyme (or as a number of ribozymes in one package), (2) reproduce, (3) evolve,) (4) create stem-loop structures as sensor-

31
reactors changing the shape or other behavior of the RNA in response to circumstances (in response to information), and (5) develop genes and other structures (tRNA, rRNA, etc.) helpful to it, as well as ultimately peptides of amino acids and proteins of polypeptides to act as needed or useful enzymes and structures. However, RNA has some disadvantages. Its single strands, though strong enough to survive with hundreds and even thousands of nucleotides, became more fragile as they grow longer, unless they fold back on themselves or pair together as double strands. In either of those cases, reproduction becomes less efficient. RNA had to be first to be successful in launching biology, but it has limitations on practical maximum size, and some of its qualities, helpful in some ways, become less needed or less effective in more advanced forms. On the other hand, no direct evidence of DNA versatility establishes its ability to excel at most of the enumerated qualities of RNA generally, but it does have three advantages: (1) it tends to be more stable, at least when protected and supported by structural proteins, so it can grow far larger while remaining effective and practical at limited tasks; (2) it can thus act as a genome quite efficiently for a larger, more complex bion; (3) at least when double, it does not form internal structures that would interfere with either reproduction or the folding and close packing needed for a very long genome; and thus (4) DNA proved more efficient as a genome and copier of RNA than RNA itself! Consequently, doubtless after various trials and errors, protobions of the fourth kingdom evolved with the ability and practice of copying their double-stranded RNA genomes into DNA and then used the DNA to make new copies of all the needed RNA molecules, including the B strand (many times over) and tRNA and rRNA, before returning to the RNA-only format when resources ran low. To do this regularly and dependably, it became necessary to refine the DNA to RNA copying, using a new DNA-to-RNA enzyme (transcriptase) and an RNADNA enzyme (known as reverse transcriptase). Both processes could happen without the enzymes but more often, more rapidly, and more accurately with them. It seems odd that any of these transition protobions of the fourth kingdom have survived to the present but they seem to have done so, since they exist today as retroviruses. Later forms have refined the process more, keeping the genes and related portions of the main strands as DNA permanently, but the RNA in its other roles (see next few steps). Perhaps it all happened quickly enough that the fourth kingdom remained in existence when a new opportunity for bions of that group opened up with the rise of cells. Or perhaps they did not survive (except in their newer and more advanced forms) but were reinvented in more recent times, when the new niche became available (as parasites), and other surviving protobions moved into the new niche. In favor of that view is the relative basic uniformity of this group of bions, which is often less true of other modern protobions. Finally, I should add that designating this group as a kingdom might be criticized by some traditional biologists on the ground that the group is too small and the members too similar to each other for such a classification. They are commonly regarded, I suppose, as a genus. On the basis of similarity they might reasonably be described as a genus. But a genus belongs in a family, a family in an order, an order in a class, a class in a phylum, a phylum in a kingdom, and a kingdom in an empire, following (mostly) traditional biological classifications. Many of these categories wind up with only one of a particular subgroup within it, either because they have never been very successful, or, tough once widespread, they may almost all have gone extinct, or again, because they have all evolved into what is now regarded as a different group. Where the facts fit such a situation, it is not relevant how many categories or individuals there are. This group of retroviruses, while small, must once have included the ancestors of all

32
the more complex forms. Further, this group is totally unique in its biological cycle. No other kingdom known follows its practice of using RNA as its normal, basic, bion strand(s), yet switches to DNA for reproducing, and the nature of the basic reproduction and of the basic nucleic acid used for it is surely the most basic characteristic of any bion. Hence, this group is logically a separate kingdom, even if that kingdom has only one genus, and this group would still be logically a separate kingdom even if it only contained one species or even one individual.

Step 47. A new specialty: mRNA.

For ordinary chemical processes other than reproduction (replication), fourth kingdom members, after coverting (copying) B strands into DNA, keep the whole of one or both of those DNA strands intact. But in early times they needed to use parts of these strands as genes to guide the creation of tRNA, rRNA, and protein molecules. The solution to this problem was to transcribe those particular parts of the BDNA molecule(s) back into RNA. That RNA molecule was the same as that particular part of the original BRNA molecule because: The BDNA molecule was originally made as the complement of the BRNA molecule and the new, partial RNA molecule would necessarily be the complement of that part of the DNA molecule. Hence, it would be back to its original form and sequence and would still work. That partial return to the earlier BRNA or bion strand, now part of the genome, is now called a messenger RNA or mRNA. It has been used in all bions that are more complex (than protobion Kingdoms 1, 2, and 3) ever since and is still used in us today. In itself this too was a revolution. The function of mRNA is to carry the code or codons message of one part of the main DNA strands instructions, i.e., one gene, to the tRNA and rRNA molecules, so that they can get from this message the information they need to build a tRNA molecule, an rRNA molecule, or a protein molecule. So this part of an old BRNA molecule, which was the essence of our earlier ancestors, has been retained in our more recent ancestors and in us. It is now the information strand mentioned in earlier steps for the development of the genetic system. But it retains only in this front-line function, no longer functioning as the huge genome as it did in the earliest bions. The genome is contained today in our chromosomes in the form of DNA (no longer as RNA). This form, the DNA genome, has remained largely unchanged in its fundamentals for nearly the last 4,000 millads (4 billion years).

Step 48. Kingdom 5: ssDNA protobions.

Some DNA protobions today have single-stranded DNA, which we could, by this step, regard as essentially only a genome. It does not retain the old RNA functions of ribozyme, reproduction starter, or even transcription starter for making enzymes or structures. All of those functions remain with RNA, as they always were. But those parts of RNA have been matched on DNA with their DNA complements, which can be matched to make their original RNA equivalents, as needed. For the DNA main strand(s), those are just more genes, i.e., messages to convey to RNA for it to effectuate. Evidently, the Kingdom 5, single-stranded DNA protobions once were Kingdom 4, transitional bions, moving from their former BRNA basic nature to DNA for reproduction, then back to RNA in times of limited resources. Either (a) they copied only their forward-oriented BRNA strand into forward-oriented DNA (as the temporary genome strand), giving them just one DNA strand, or (b) they originally copied both RNA strands into DNA strands but later, somehow,

33
lost one. Some of these still exist today. The only ones so far discovered are parasites (viruses). No members of this fifth kingdom appear to have advanced any farther than that.

Step 49. Kingdom 6: dsDNA

Some members of Kingdom 4s transitional protobions also refined and economized their operating system by keeping the DNA complements of both of the former BRNA strands as their only genome, after while never retranscribing them back into the whole RNA strands again. This step established the sixth protobion kingdom, the permanent specialization or division of labor between RNA and DNA. Ever afterward, DNA was largely the genome, nothing else was part of the genome, and RNA was left with its other former functions. That remained true ever afterward for protobion Kingdom 6 and for every newer kind of bion after that, except those that did not get through the Kingdom 4 stage. So that division of labor between DNA and several special-function RNAs, as well as the entire genetic system, has been inherited and kept by all cellular bions including us, all of which came later. Step 50. Looking back, forward, and deeper. a. Achievements and kingdoms of protobions thus far We can see, then, that biology started with the first bion, an RNA molecule, that as the complement to another RNA molecule which served as a template. After this first form was improved by a starter section for reproduction, or more properly replication, multiple other improvements followed in rapid succession, including stem-loop structures, ribozyme functions of the RNA strand, special structures like tRNA, rRNA, mRNA, capsids, docking sites on the main BRNA, development of the entire genetic system (including its codons, genes, peptides, and proteins as enzymes and structures or parts of structure), switch from single to double strands, and then from RNA to double-stranded DNA for the permanent genome. This progress included the establishment of six separate protobion kingdoms, with our earliest ancestors passing through five of them in succession (and all these kingdoms remain in existence today, though in much different circumstances than in ancient times): Kingdom 1. Kingdom 2. Kingdom 3. Kingdom 4. Kingdom 5. Kingdom 6. Bioids Single-stranded BRNA molecules Double-stranded BRNA molecules Genome-molecule switchers Single-stranded DNA (a dead end) Double-stranded DNA (pioneers)

These six kingdoms composed, and still compose today, the protobion empire, or first bions. This first realm, or empire, the protobions, could also be called the non-cellular or precellular bions. They were and those still in this category remain without the cell membrane that is standard for all categories of biota that developed later. Our ancestors passed through each of these stages and thus, for a time, were members of each of those ancient kingdoms (except, perhaps, Kingdom 5). We are, as has been mentioned, cellular bions, made up of cells, each of which has the features mentioned in this volume so far, except in kingdom five, including DNA and RNA, genes and the whole genetic system with its specialized molecules and organelles (little organs), ribozymes, both enzymatic

34
and structural peptides and proteins, etc. But this narrative is not yet at the point where we can begin to talk about cellular bions. Much more is yet to come. b. Looking forward. After these first steps described above, many more had to come before formation of the first successful cells. Cells have had great success on Earth, but the very simplest known (and not known for long) have about 500 genes. Those features described above remained far below those in the later stages of biology on Earth as described so far. That is a good sign that cells cannot have existed on this with what we have described so far. Few protobions remain with more than a few hundred genes but a few have been reported which do have nearly 500.52
Genome sizes vary in viruses, with the smallest occurring in ssDNA circoviruses, coding for two proteins and containing 2 kilobases; the largest occur in mimiviruses which contain over 1.2 megabases and code for over a thousand proteins (Wikipedia article Virus Genome citing Van Etten, J.L., L.C. Lane, and D.D. Dunigan. 2010. DNA viruses: the really big ones (giruses) in Annual Review of Microbiology 64: 83-99. Doi.101146/annurev.micro.112408.134338. PMID 20690825). The smallest genome in a cell that I have seen referenced is that of a bacterium, Carsonella ruddii, the genome of which contains 159,662 base pairs of DNA and 182 protein-coding genes (Ball, P. 2006. 2006. Smallest genome clocks in at 182 genes, in Nature News (12 October 2006). Doi:10.1038/news061009-10, citing Nakabachi, A., A. Yamashita, H. Toh, J. Ishikawa, H.E. Dunbar, N.A. Moran, and M. Hattori. 2006. The 169 -Kilobase Genome of the Bacterial Endosymbiont Carsonella, in Science 314: 267. Doi:10.1126/scinece.1134196. Also citing Prez-Brocal V., R. Gil, S. Ramos, A. Lamelas, M. Postigo, J.M. Michelena, F.J. Silva, A. Moya, and A. Latorre. 2006. A Small Microbial Genome: The End of a Long Symbiotic Relationship? in Science 314: 312-313. Doi:10.1126/science.1130441. In contrast, the human genome contains around 3 billion base pairs.
52

That approximate uniformity of numbers of genes in the most complex protobions and the least complex cells strongly implies that such number marks a biological frontier between the maximum potential of the protobion structure and system and the minimum possibility of the cellular structure and system. That frontier must have been reached (and crossed) by not long after 4,000 millads ago (4 Ga), certainly no later than 3,900 millads (3.9 Ga). The numbers of genes in these two empires (protobions and the first cellular bions) were arrived at independently by two independent groups of scientists (researchers of modern forms of protobions called viruses and different researchers of the tiniest, modern, cellular bions called mycoplasma)53, neither of which, judging by their publications, would have accepted the basic thesis of this volume. That set of facts adds considerable weight to their evidence on the fact of this important biological boundary, and its prehistoric crossing before the second oldest rocks accessed on Earth by geologists were laid down by geological processes (4 to 3.9 billion years ago), because those rocks contain organic deposits clearly laid down by populous and highly successful biota able to leave extensive traces of biological activity. These deposits show the single-type chirality unique to biological chemistry.
53

Mycoplasmas have small genomes of 0.58-1.38 megabases (Wikipedia article Mycoplasma citing, among others, Fraser, C.M., J.D. gocayne, O. White, M.D. Adams, R.A. Clayton, R.D. Fleischmann, C.J. Bult, A.R. Kerlavage, G. Sutton, J.M. Kelley, R.D. Fritchman, J.F. Weidman, K.V. Small, M. Sandusky, J. Fuhrmann, D. Nguyen, T.R. Utterback, D.M. Saudek, C.A. Phillips, J.M. Merrick, J.F. Tomb, B.A. Dougherty, K.F. Bott, P.C. Hu, T.S. Lucier, S.N. Peterson, H.O. Smith, C.A. Hutchison, and J.C. Venter. 1995. The minimal gene complement of Mycoplasma genitalium, in Science 270 (5235): 397-403. Doi:10.1126/science.270.5235.397. PMID 7569993).

35

Not all of the genes of the most complex protobions surviving to today have been completely studied, but enough has been done to show that most are devoted to adapting to their present environment, which mainly involves coping effectively with cellular biota, mostly bacteria, which they parasitize. Genes of this specific type obviously cannot have evolved before the existence of cellular bions. But the coincidence of the two genome sizes rarely more than about 500 genes in protobions and rarely fewer than that number in cellular bions implies that protobions have a 500-gene effective potential. Before even the possibility of, much less the need for, parasitizing cellular biota, what would those last few hundred potential genes have been? Most reasonably, the basic genes needed for the time, place, and circumstances in which they did find themselves. So then what did our Kingdom 6 protobion ancestors still need before the cellular stage? We cannot discern the answer from surviving protobions because their other genes largely related to their present circumstances (i.e., the existence of cellular bions that did not exist when this kingdom of protobions evolved), very different from the ancient setting. Hence we must consider what all the simplest cells have, and must have, to become what they are. Before embarking on the next 50 steps in search of the answer to that question, let us look deeper and more broadly. c. What do biologists generally tend to say about the distinctions between organisms and other objects? First, some use the term life, while others avoid it for living things or alive. As chapter C stated, these are words for unclear, emotionally charged, and, to some degree, obviously mistaken, ideas. A better term is our bion, something composed of or containing a nucleic acid molecule, probably at least 100+ nucleotides long, which owes its existence to having been formed by nucleic acid construction as the chemical complement of an existing nucleic acid template, and which continues to have the potential to perform the functions with which it was formed. This is quite precise, inclusive of all biota, and exclusive, so far, of non-biological things. (Some engineers are trying to destroy this distinction by using nucleic acid for making atom-sized machines54 and thereby acquiring large amounts of money. This ought to be strictly and firmly outlawed. The health consequences will make Jurassic Park look like a sunny walk. Humanity will deeply regret the consequences if such products are introduced on a significant scale into our environment.)
54

The author speaks here of nanorobotics, an emerging technology field, in which tiny machines or robots -9 are constructed with dimensions measured in nanometers (one nanometer is 10 meters). These devices are also referred to as nanobots, nanoids, nanites, nanomachines, and nanomites. Although in the developmental stages, these tiny machines may potentially be used in the field of medicine to perform tasks on the cellular level. Current drug delivery methods generally do not target particular cells, which often leads to unpleasant side effects, particular in cancer treatment. As the Wikipedia article Nanorobotics notes, researchers have built some nano-devices filled with a chemotherapy medication, in the form of RNA strands that are naturally attracted to cancer cells. Such a device seeks out a cancer cell, attaches to it, and released the drug directly into that cell. Ideally, such a method will kill cancer cells without also attacking healthy cells and so avoid the severe negative side effects typical of current delivery systems (See, e.g., Debjit bhowmik, C. R.M. Chandira, and B. Jayakar. 2009. Role of nanotechnology in novel drug delivery system, in Journal of Pharmaceutical Science and Technology 1 (1): 20-35). The authors fears seem to be based on the idea that such devices would replicate and outstrip their original purposes, perhaps even mutating into virulent forms, with untold consequences.

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Researchers in this field are aware of this potential problem, however, and are likely to either prevent or severely limit self-replication. The warning the author gives refers to the movie, Jurassic Park, based on the book by Michael Crichton (2012. Ballantine Books, originally published in 1990), in which dinosaurs are brought to life in modern times by joining DNA remnants found in insects preserved in amber, amplified with frog DNA, with eggs hatched in a lab, with ultimately disastrous results.

Among criteria for bions suggested by various textbooks on general biology, the following are included: (1) Reproduction. Clearly, no spies can last unless a significant number of its members can reproduce, so, as suggested so far in this project, this is part of the defining characteristic, but an individual bion remains such even if, for whatever reason, it does not reproduce or has not yet done so. Many individuals cannot or, even if theoretically capable, do not. So the definition must be the result of such reproduction, not the capacity or necessity to repeat it. Also, reproduction, in itself, is not enough. It must be, at least on Earth, by the process defined above (by nucleic acid replication based on a template). Fires, ice cracks, microscopic drops of oil that are continually fed, etc., can reproduce approximate copies of themselves, but they are not biological. They are not bions. (2) Definite organization. Certainly individual bions have a definite organization, but so do many other things, so that much is not distinctive. Do all bions have a definite organization that is common to them? Superficially, no, they may be round as a ball (coccus bacterium), tubular like a worm or snake, indefinitely branching like a bush or tree, stool-shaped (mushroom), etc. But all have (a) a basic, controlling, nucleic-acid dominant section, and (b) a genetic system including the standard code or a close approximation, with all the supporting mechanisms previously described. Most also have genes making possible the creation of specific proteins and their shorter sister, the peptides, and use this capacity to make such products for the benfit of the bion. (3) Irritability. ? Biology texts often say that all their subjects are able to detect and react to various features of the environment, which may include atoms, molecules, other biota, even themselves, temperature, sunlight, wind, dust, water, pressure, pain, etc. This is widely true, but, as we have seen, it is not, in fact, universal in biology. The simplest protobions often lack it (bioids and some ssBRNAs), the spores of bacteria seem to be inert to any influence except return of the favorable conditions in which they can reanimate as bacteria again (spores exist as an emergency condition to ride out disastrous conditions), and even those bions which do have the ability to detect and react to environmental or internal conditions vary widely in the nature of the conditions to which they can respond, the number of such conditions, and the degree of specificity of the detection and response. This quality may have increased and improved among protobions after what we have previously described. (4) Feeding. One general biology text lists feeding a universal bionic characteristic. By that the author seemed to mean taking substances for incorporation into its own organization.

37
This is generally true of animals, most protists (single-celled bions with cell nuclei like ours), as well as plants, which take in small molecules through their roots and leaves, prokaryotes (small, cellular bions which absorb small molecules through special molecular tubes or gates in their cell membranes), and fungi (in the manner of prokaryotes). But it is not true of the protobions to the point that we have covered them so far, unless we count incorporation of extra nucleotides. This is thus a characteristic which we might expect to have developed further, later than what we have described so far, but before cells arose, since it seems universal of cellular biota. (5) Use of adenine triphosphate for energy flow. We have already taken note of the need for input of energy for some biochemical processes, particularly the building of organic structures (molecules), and that the presence of phosphate ions and the four early nucleotides in fact assisted protobions near geothermal vents and plumes in the period covered so far. Adenine triphosphate is simply one of those early nucleotides, with two extra phosphate ions attached to its normal one. It was not, and is not now, the only one. These free-floating nucleotides performed that service within particularly enriched portions of ancient, pre-cellular Ocean, during the period that we have covered, and continued to do so to the present in all other familiar biota, so this characteristic does seem to be as universal among bions as nucleic-acid template replication. (6) Inheritance of size and form. Just as being a bion is inheriting (approximately) a size and shape of something similar in reproduction, it follows that, in general, bions tend to inherit at least a size, shape, and organization that resembles the parent, within broad limits. For RNA molecules, the approximation is fairly similar, and it is for single cells, although variation, mutation, and evolution are ever present. Multicellular bions, however, may be born much smaller than the adult that gave them birth, even though their individual cells are quite similar to those of the parents. Also, wide variation in the appearance and activity of multicellular bions of the same lineage may differ widely in accordance with age, or even generation. So it is the potential size, shape, and organization that is inherited, not necessarily the particular one taken at a given time. With these cautions, the idea seems fairly reasonable, but not always definitive or helpful. (7) Predetermined lifespan. This characteristic applies to some bions, but not nearly to all, or even to most. It does not apply to either protobions or to prokaryotes, nor does it apply to eukaryotic singlecelled bions, and those three categories certainly include most bions on Earth. This suggested universal characteristic of bions is clearly false and does not suggest anything pertinent to the rest of this volume. (8) Ability to move as a response to the environment. This, too, is false. Some bions have such an ability. Animals normally can move. Plants can move parts of themselves very slowly. Some fungi make various kinds of moves, mostly not immediately visible. Protists with flagella or cilia can, as can amoeboid protists, and some spiral or flagellated prokaryotes can, but many others cannot. Of course, we are aware that no protobions have been shown to move much, although there is one category of such bions that can make very tiny, functional movements. It is

38
probably not necessary for us to consider this ability in this volume. The only movement involved in this volume is the passive type due to environmental water moving. (9) Growing. ? Some biology texts list growing as universal biological phenomenon. It is certainly widespread. Prokaryotes grow modestly from the time one cell splits into two (each half as big as the parent), until those two grow back to a size permitting them to split again (a half hour for some well-fed bacteria). The protobions studied so far generally do not grow, although in their early phase they probably sometimes added one or more additional nucleotides, or perhaps even joined each other end to end, producing a kind of growth. But otherwise, what is the advantage of growth? For animals, it permits a parent to produce more offspring and for some to care for them in their early life. For plants, it assists dissemination over a wider area. It seems incompatible with the nature of protobions. The only universal advantage would be to add material for use in producing new bions or for supplying energy and structural repair and rebuilding. The protobions that we have studied do not build up stores of anything, but prokaryotes do, within limits. We shall have to consider whether it was feasible and advantageous for our later protobion ancestors to start that practice. (10) Life is a condition of protoplasm. This astonishing statement actually appears in a modern biology textbook. This is backward, upside down, and absurd nonsense. Protoplasm is a vague word invented to identify the unknown contents of a cell. Today, the contents of many cells are fairly well known. They are many and their properties are not particularly similar. The only things they have in common, except for their location, is that they interact in various ways to keep the system going. In this sens, biology is a system, but it is not just any system, but rather a particular system built from and based on the protobions that we have been meeting. We could say, closer to truth, that Life is a particular system characterized by, starting from, and built upon the reproduction accomplished by nucleic acids, using a nucleic acid template process. But the difficulty of using the term life has already been discussed. At best, the statement quoted as the heading of this paragraph conveys no real information and thus is worthless. At worst, it is misleading. (11) Display of metabolism. ? Many biologists assume that protobions cannot be alive because metabolism is universal among living things. This, of course, is circular reasoning, arising from the fact that those bions which do display metabolism were known before the existence of protobions was discovered. Clearly, the conclusion drawn is unsound, for reasons recited previously, so I will repeat them here. Still, metabolism is widespread. We therefore should consider whether it needed to arise to reach the stage of cellular life, is so in what sequence, and how it did so. We shall deal with that later. A word more should be said. What is metabolism? This is a word for a number of related changes, which had to arise piecemeal. The two main parts are anabolism and catabolism. The first is building larger molecules from smaller ones, or from smaller parts of other large molecules. This could be making new bions (reproduction) which, as we have seen, protobions have consistently done. Anabolism could also be building molecules for storage, either of structural material or material useful as an energy

39
source. The rise of metabolism is there part of the same processes that are involved in energy flow, feeding, growing, etc., mentioned above.

(12) Some additional general characteristics of biology have been prominently asserted in textbooks: (a) No [bion] can exist indefinitely We have dealt with that error under paragraph (7) on the predetermined lifespan. (b) A bion cant just exist, but has to be engaged in constant activity Technically, this may not be totally wrong, but it is quite misleading. The statement went on to deny the possibility of suspended animation. It seems true enough for animals, appears so for plants, and may be for some other biota, but the protobions that we have considered up to now are essentially inactive when not preparing to reproduce or doing so, and the spores of bacteria are quite close to inactive. They are bacteria which, facing adverse conditions, squeezed out their own water, limiting their biological activity to a minimum, simply waiting out the bad times (or dying). No activity has been reported as detectable during this time, even though they spring up again after this dormancy. There probably is a difference between dormancy and death in this context, but any activity is slight. (13) Excretion. Excretion has been listed in biology textbooks as a universal characteristic of biology. Three comments apply. First, excretion is not entirely unique to biology. Some other systems, in effect, also excrete substances not used. In fact, many chemical systems rid themselves of material resulting from a chemical interaction, which therefore no longer participates, as in some examples early in this volume. Second, the protobions already considered did not have anything to excrete and did not excrete anything. Neither do their modern descendants which remained in that biological empire. Even some bacteria apparently are unable to excrete certain wastes, memely keeping them as inclusions inside themselves. (The same may be true of other prokaryotes, but the other major kingdom of them, the archaea, are not yet well studied and reported because of their remote and difficult environments.) This, excretion would seem necessarily to depend on feeding, metabolism, and the amount and nature of any wastes collected in those processes, so it will have to be considered in the remainder of this volume. It should be noted that, among the 13 paragraphs of this list, those supposed universals where the paragraph is followed by a check mark () are accepted as sound. Those followed by nothing are rejected as unsound. Those followed by a question mark (?) will be examined as potential directions of evolutionary changes occurring between the time of the establishment of Kingdom 6 above and the time of the arrival of cellular life. Let us proceed, therefore, to consider what further progress may have been accomplished by our ancestors in the sixth kingdom of probions beyond those mentioned earlier.

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A spherical bion of Kingdom 4, an oncornavirus (from the authors hand drawing). Black lines are receptors. Red circle is the lipid layer, derived from the host cell. Orange hexagon is the capsid. RNA is green. Small blue circles represent an enzyme, in this case protease. The green circles at the ends of the RNA represent reverse transcriptase. The lentivirus is similar, but the capsid is more or less a teardrop shape rather than a hexagon and the RNA is in two separate segments.

Partial image of the helical tobacco mosaic virus (from Wikimedia Commons, article Virus).

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Structure of the T4 bacteriophage, a virus that infects bacteria (from Wikimedia Commons, article Bacteriophage, section Virion Assembly). While the author of this work uses the term virion to mean a very simple virus, the usual meaning is the complete virus, both the capsid (outer shell) and the nucleic acid. When it infects a cell, the the virus separates these two components. The capsid remains outside the cell while the nucleic acid enters the cell, infecting it.

Depiction of the T4 bacteriophage infecting a bacterium (from Wikimedia Commons, article Bacteriophage).

42 Chapter I. Steps 51-60 Refining the Genetic System

Step 51. DNA, Thymine, and Amylation.
In considering the air of the early Earth (after elimination of any large excess of hydrogen during the first 50 millads of high surface heat and a massive collision with another planet), we met the four most common elements, the atoms of hydrogen, oxygen, carbon, and nitrogen, some of their many compounds, and various molecules made of them, such as ammonia (NH3), water (H2O), aldehydes (such as CH2O) and sugars (C4-5O), and methane (CH4). Later, with respect to compounds like amino acids and heterocyclic organic bases, we noted that all four of those atoms appear in various parts called functional groups, such as amine groups from ammonia (-NH2), carboxylic acid groups from organic acids (-COOH), etc. Another such group, from methane, is called a methyl group (-CH3). Next, when we discussed the organic bases in RNA, one of the pyrimidines, a single ring of four carbon atoms and two nitrogen atoms, and with attached (non-ring) oxygen atoms bound to the ring-carbon atoms on either side of one of the nitrogen atoms, was uracil. Later, when DNA was substituted for RNA as the persistent genetic material, it became necessary to change uracil a bit. Its role in DNA is exactly like that of the old uracil in RNA, where it is still the standard molecule for that role. It is not clear why the loss of one oxygen atom from the sugar component of RNA, which makes it into DNA, should require such a change in uracil, but it happened, and is consistently followed in biological circumstances now. Perhaps a change occurred in the amount of some element, the temperature or chemical balance in the environment, or some other factor that made putting one oxygen atom into ribose less easy than before. More likely, biology had just progressed to the point in complexity or other problems that the advantages of a permanent DNA genome kept away from contact with the outside world simply became great enough to outweigh the early advantages of starting with RNA as a genome along with using parts of itself as ribozymes, sensors, reproduction starters, genetic-system parts, etc. In other words, Kingdom 6 double-stranded DNA protobions had become complex enough to be forced into the first of many modifications toward a separation of functions into different, more specialized structural modules. As said in chapter C, modular specializations are typical of complex structures. Certainly biology is complex enough for that. It did not start out that complex. Compare Kingdoms 1 and 2, comprising bions that were simply single strands of RNA, despite the numerous preceding chemical changes, assisted in some cases by catalysts and early fortuitously (and non-biologically) formed enzymes, that later brought together a phosphate ion, a ribose sugar, and an organic base into a nucleotide, and hundreds of nucleotides into the RNA strand. Yet at some point, growing complexity was sure to reach the point where modular separation was necessary. In the case of bions, it first happened with the specialized parts of the genetic system, then with the transitional protobions of Kingdom 4, and fully in Kingdom 6 with the permanent dsDNA genome. What was the specific change in uracil to complete this addition of that module? That change was the addition of one methyl group (in place of a hydrogen atom) bound to the ringcarbon atom opposite the one holding an oxygen atom (see figure below). Uracil with this change is actually called by a different name, thymine. So, just as the single-ring (pyrimidine)

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uracil pairs with the two-ring (purine) adenine as a complement in RNA, the single-ring (pyrimidine) thymine pairs with the two-ring (purine) adenine as a complement in DNA.

Ball-and-stick uracil molecule (from Wikimedia Commons). Red balls represent oxygen atoms. Blue balls are nitrogen atoms (with white hydrogen atoms linked to them). Black balls are carbon atoms (with white oxygen atoms linked to them).

Ball-and-stick thymine molecule (from Wikimedia Commons). Colors represent the same types of atoms described in the previous figure. The rings in uracil and thymine are identical, but a methyl group is attached to the carbon atom of thymine on the side of the ring opposite to that between the nitrogen atoms.

(Actually, adding a methyl group to one of the organic bases occurs in various biological contexts, sometimes conveying a particular refinement in meaning. In this case, it distinguishes this new form of single-ring base for complementing adenine as being in DNA, part of the genome, rather than part of the RNA information strand still used for structuring protein products -- called mRNA -- and some other purposes.)

Step 52. Protection, inclusions, and support.

a. Protection. As previously noted, once the multi-part genetic system neared completion, it was able to craft a capsid to protect the nucleic acid, including its starter, its growing genome, and other parts from chemical damage, physical disruption, incorporation into other systems and entities, and other dangers. With time, these capsids became widespread among protobions, varied in details and complexity, and still later some became multilayered structures in protobions of Kingdom 6 (dsBDNA), with each layer providing a different kind of protection from the others. These changes in protection for the genome conferred at least four changes to the bion.

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(1) First, of course, the mere formation of a capsid of any type adds a further part or module to the protobion, so henceforth those with such a feature had the following modules: (a) The RNA starter; (b) tRNA, rRNA, and mRNA parts of the genetic system; (c) RNA sensor-reactors; (d) A few surviving RNA ribozymes; (e) The DNA genome, in two strands complementary to each other and bound to each other by hydrogen bonds in the famous double helix that is fundamental to all the descendants of one of the members of this kingdom including us; and (f) The capsid itself. (2) Second, protecting the genome from chemical and some physical damage reduced the number of mutations otherwise caused by such damage, thus improving the accuracy and efficiency of reproduction, as the growing genome made such accuracy ever more important. (3) Third, the existence of the capsid itself, of whatever size, increased the size of the protobion. (4) Finally, the capsid enabled these bions to contain additional useful molecules and other parts. b. Inclusions. Next, taking advantage of this new ability to contain, some dsBDNA did place additional molecules inside the containment space. In one case, it was an enzyme useful for reproduction, thus making it immediately available and quickly usable at the time of replication. The result was faster replication and a further reduction in the likelihood of mishaps during the process, a considerable source of damaging mutations. The reduction of the mutation rate meant (and means today) more accurate genomes with few losses of useful functions. The disadvantage would be fewer new and useful mutations due to the slowing rate of mutation overall. With long genomes, though, the balance of effects was favorable. c. Support. Another kind of inclusion within the capsid, aside from the dsBDNA and any reserve supply of enzyme, was in some cases structural proteins of various kinds to help the DNA helix retain its proper structure and spatial arrangement, further reducing the danger of breaks and tangles that might interfere with proper DNA function. Not every protobion has this feature, but dsDNA bions of the sixth kingdom of the protobion empire commonly do. These proteins vary, but one of them may have been an ancestor of the histone proteins, which later played a similar role in the next two biological empires to come, including us. These have been pictured in at least one source as roughly spherical structural proteins filling up indentations (called grooves) occurring in the DNA helix as a result of its helical, twisted, or spiral staircase structure. Over time, these proteins seem to have acquired additional characteristics and functions and today, in us, are not merely passive spindles (or spools) around which the DNA helix can wrap, but active guides and message carriers for messages initially brought by short, single-stranded RNA molecules from genes in other parts of the DNA strands.

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Step 53. Micro-environments.

Up to now, all that has been said about the geographic origin of protobions has been that the four early-air elements were necessary and contributed to both the preliminary steps of small-molecule pre-biological synthesis of biologically important molecules in the air and Ocean, and that the next steps, aside from metal-catalyst effects, had to occur in the vicinity of the geothermal vents and plumes that occur in the deep Ocean along the ridges where recycled molten magma from below wells up through the Ocean floor, powering continental drift. Although some writers reject such a location on various grounds, including the usually short lives of modern biological RNA in high temperatures, nevertheless such a general location seems very convincing to me. It provides a continuous and generous supply of phosphates, which are crucial to biology in two fundamental ways. First, every nucleotide of every nucleic acid must contain a phosphate ion. As emphasized before, biology starts with nucleic-acid template replication. Even if other molecules are necessary and may be made in the air or Ocean generally, they can easily reach the ranges where geothermal vents abound (these areas trace their ways around the Earths Ocean bottom), and must have done so to progress further to biological status. Thus, they are all connected, providing both a way for biological progress to spread easily, and a landing place for any early bions swept away from one such environment to another Ocean currents, meteorite falls into the Ocean, etc. Second, besides the elements needed, a ready and lasting supply of chemically available energy is necessary to fuel the making or synthesis of complex biological molecules on a consistent basis, and only in such areas is that consistently available. It comes, for our concerns, primarily in the form of nucleotides with extra phosphate ions attached to them. That is one reason why nucleic acids became the foundation of biology and brought it into being. Thus, as mentioned earlier, each nucleotide can, and commonly does (along the deepOcean magma ridges), appear with these extra phosphate ions, as a provider of energy, a potential joiner of nucleic acid chains, a builder of ever bigger biological structures, and a crucial ally in the building of almost all biological molecules. As we know, nucleotides contain a phosphate ion at one end, pentose sugar in the middle as the link to the third part, and that third part is the organic base. (Thus the sugar in the middle has a bond to each of its partners in the nucleotide, one with the phosphate and one with the base.) If such a nucleotide joins a nucleic-acid strand, it does so by binding the sugar at a third point to the phosphate ion of another nucleotide, and its own phosphate ion is joined by the pentose (ribose or deoxyribose) of a different nucleotide. In each such joining step, the energy price of being able to join is the loss of one or two of the extra phosphate ions attached to still another point on the pentose sugar. So that sugar must have attached to it at all times the phosphate ion that is part of the nucleotide and must also have one, or more often two, phosphate ions in addition, which are lost or paid to get the chemical energy necessary for building a bigger molecule. (That remains true today throughout biology, including within every cell of our bodies. We carry an equivalent of that ancient, deep-Ocean environmental system within us, modified to fit todays circumstances.) (We might note that a nucleotide with only its one necessary phosphate ion is called a monophosphate. If it has one extra phosphate ion, it is a diphosphate and if it has two extra phosphates, it is a triphosphate. For example, we read of many biochemical interactions driven (energized) by adenine (others by guanine) triphosphate, where one phosphate ion is lost in

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providing the needed energy for the reaction, changing the energizing molecule to adenine (or guanine) diphosphate. Where the process is building a new strand of RNA or DNA, both extra phosphate ions are lost and the remaining monophosphate is incorporated into the growing nucleic acid strand as the next member nucleotide. The same is true of all incorporated nucleotides, although use of the others merely to provide energy is less frequent. Normally only one kind of such nucleotide triphosphate (such as adenine) is used just for energy in one kind of chemical reaction. Other kinds are used in other reactions, but all are used in forming nucleic-acid strands.) Besides those considerations, sulfur, metal sulfides, other metal ions, and metalliccontaining surfaces also exist in the same general areas, also playing vital roles in the origin of biology, especially in making certain crucial chemical bonds and in various catalytic roles that appear necessary for all biology, though not yet incorporated into individual bions during the period mentioned so far. After all, in that open range environment of the Ocean, incorporation of all elements needed only at intervals was not yet necessary. Even in our own time, protobions still function in such areas, in numbers an order of magnitude greater than all other biota in the area combined. As to the temperature problems for RNA, three observations are pertinent. The first is that the high pressures characteristic of these geographic areas largely negate the temperature problem. The pressures there keep the water liquid (preventing boiling) and related chemical problems. The second observation is that RNA in cells today is basically designed to disintegrate rapidly so that it can be quickly recycled after use. The opposite would have tended to be true of early protobions which, as mentioned, could help stabilize themselves by various shaping techniques, such as sections of doubling back with stretches of complementary nucleotides. Finally, as noted in the prior paragraph, protobions today survive quite well in precisely the environment we are discussing and easily outnumber all other biota in the area. Much other life flourishes there too, because of the flow of both chemical energy and abundant nutrients brought up by the geothermal plumes. Beyond these generalities, though, there has been no mention here of the precise sites of the early protobions within the geothermal areas. We have no direct evidence on the point. These areas have not been thoroughly studied because of their remoteness, high temperatures and pressures, and Oceanic realms, all presenting great obstacles and little enticement for humans. I would infer a high probability that many protobions in early days and today reside or resided in cracks, fissures and pores (the spaces between sand or clay grains) in the Ocean floor, in and for some distance around (or rather on all sides of) the ridges and their vents and plumes. Recent studies show that prions (defective, misfolded proteins that can influence similar properly folded proteins to change to the misfolded form) attach themselves to negative electric charges in clay particles by means of their own positive electric charges so that the clay cannot be washed clean of them with mere water. Modern protobions (viruses) also carry positive charges so they could have used the same technique. Some can be found in open water. Some others are likely to be on surfaces of various kinds and those that stayed there over time may have found ways to attach themselves. Also, some likely landed, gathered, stayed, and reproduced on floating objects stirred up by water movements or kept floating by small size the colloid particles mentioned earlier. In all of these environments, the early protobions surely multiplied, formed communities which evolved over time to enough variation for them to help each other by using abilities unique to themselves in the communities, thus sharing the benefits. For example, one might make tRNA

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of one or two kinds, another might make others, and all might benefit from the joint and diversified supply. Such biological communities can and do occur, increasing the survival prospects of all members.

Step 54. Precision versus mutation.

As mentioned previously, the first bions had great potential but no actual abilities except passively to be used by passing nucleotides as a template for building chemical, physical, and spatial complements of their own simple RNA structures. That potential was relatively rapidly developed because of a high mutation rate, giving the pressures on survival and reproduction a wide scope for weeding out defects in favor of more successful individuals and communities. As population, variety, and complexity steadily grew, the combination led to increasing limitations on the ability of protobions to grow bigger, to main their increasingly complex systems successfully, and especially to increase their growing genomes. Several steps then occurred, leading to reductions in mishaps and improvements in the precision of reproduction. The first of these, already mentioned, was the development of the genetic system to the point at which capsids could be built around the naked, single-stranded RNA bions. This achievement could protect the crucial part of the bion from most chemical and few physical dangers. Strands became less likely to be broken by physical water movements, especially the violent ones which might occur if a large meteorite arrived too near (possibly significant until 4.2 billion years ago). A capsid might even delay heat damage, although that does not seem to have constituted a significant problem. Still, replication required coming out of the capsid to be accessible to free-floating nucleotide triphosphates, necessary for building a new RNA strand. Improving enzymes could both speed the process (reducing exposure time) and improve the accuracy of matching the template. A third step of this kind, the formation of double-stranded RNA bions, helped to keep the ancestral sequence of nucleotides intact and consistent, by (1) increasing molecular stability, (2) reducing the exposed surface, and (3) keeping each nucleotide almost continuously bound to its complement. This last feature encouraged each of the two bound nucleotides to remain what it was. A problem arises, however, in replication, because of resistance to separation of the strands, necessary before that process, to allow each strand to build a new partner. The further progress to transitional and finally permanent double-stranded DNA genomes greatly eased the separation process, and also improved the mutation rate in important ways, both from damage and from miscopying. That major set of events changed the biological world. The fourth step in reducing mutations and copying errors, then, was the switch to permanent DNA genomes. DNA is more stable than RNA, so less likely to change/mutate, less likely to be damaged by chemical attack or physical agitation. Further, it is strong enough to accommodate much longer strands and thus more genes with the resultant sophisticated adaptations. The double strands are also more readily separated from each other for reproductive purposes than is RNA. Further, by separating the permanent genome DNA from the new, single-purpose mRNA for attachment sites for tRNA and rRNA molecules in building proteins, the new system reduced contact between the genome and the outside world, also reducing the risk of changes. By having double strands of DNA, here as with dsRNA, each strand tends to keep the other faithful to its prior structure and composition.

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As we know, the dsDNA bions also generally have their protecting capsids, often with multiple levels of enclosure, sometimes with each composed of a different material, increasing the range of dangers protected against. All of these factors combined to reduce mutations from damage significantly. In addition, genes have been steadily refined so that an army of enzymes can keep the construction of both RNA and DNA very precise, reducing both genetic mutations and defective proteins (of course, these are not entirely eliminated; no system works at 100% efficiency and accuracy in reality, but both genetic reproduction and protein production are extremely accurate now, and has been for a considerable time). Even when all these factors fail and errors do occur in the DNA, cells generally now have ways to repair some of these. Modern protobions generally do not. It is not entirely clear yet whether this improvement occurred before or after the first bionic cell arose, but it seems fairly persuasive the event must have occurred to that time. Because this improvement is too important to omit from our story and this volume emphasizes chemistry more than later volumes, here seems the best place to explain it. DNA repair systems can correct a few kinds of problems, including what are called nicks, breaks, and wrong nucleotides. A nick in this sense is a missing nucleotide in one DNA strand but not the other. Because the two strands are held together by hydrogen bonds (weka individually, but fairly strong collectively over a strand), nucleotide for nucleotide, if one nucleotide on one strand is missing, that strand will not ordinarily heal themselves immediately, because that would require a large number of nucleotides moving one space over, no longer leaving them in position to retain their hydrogen bonds. Instead, they retain their places, leaving a hole or break between the two parts of the strand. Special enzymes can recognize such a situation and insert into the breach the nucleotide that complements the one opposite to it in the other strand. Only one of the standard four nucleotide types used in DNA will normally fit in that place. The breach in the strand is then healed again by enzyme action. The enzymes are able to do this because (a) only one nucleotide will properly fit and (b) the opposite nucleotide, which must be complemented, is still present and hence identifiable. In a similar way, the current system can repair a wrong nucleotide, which to biochemists means an instance in which one nucleotide does not match (complement) the nucleotide that it should match in the opposite chain. This situation differs from the first in two crucial ways. First, of course, the repair has to replace something that is present, not fill in something that is absent. The appropriate enzyme must therefore cut out the wrong nucleotide, removing it from the strand, in effect creating a nick. Yet, second, how can the enzyme system choose which of two non-matching nucleotides to cut out, when each should (but does not) complement the other? The choice can only be made when one strand is the old, existing one, being used as a template against which to build a new strand. In such a case, the nucleotide in the old, template strand is taken as the guide. The nucleotide opposite it in the strand is removed, a match for the nucleotide in the old strand is inserted in place of the removed one, and the strand is healed again. In the remaining case, a break, nicks arise on both strands, matching empty space with empty space. Thus, in this case, unlike the previous two, there is no remaining nucleotide in the right position to match/complement, so there is no way for the system to know what belongs in either of the two matching empty spaces. Now such a situation has two imaginable responses. Should the system merely reattach what is left (refasten the two halves of each strand)? The advantage of that approach is that later attempts either to replicate, making a new DNA strand, or to create an mRNA to build a protein, would fail if the two fragments remained

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separated. In the second case (making mRNA) the appropriate enzymes and nucleotides would find the pertinent starting place and normally proceed, one nucleotide at a time, until reaching a stop code or the end of the DNA strand. If the breach remained, no stop code would be found, and everything on the remaining fragment would be ignored. That would be unacceptable. On the other hand, we have noticed that the code (or codon) for a tRNA docking site on an mRNA molecule consists of three nucleotides. The system has no equivalent of punctuation except for the start point and the stop. Between those two markers, each nucleotide comes next to those before and after it. The result of that is that reading the nucleotides must start at the correct place, take them one at a time but interpret them three at a time for a docking site or codon, and go on to the next nucleotide. If the two fragments of the DNA are merely reattached to each other directly, with one former nucleotide now missing, everything after that point will be read wrongly both within the coding for one mRNA and throughout replication, because the reading frame will wrong. For example, suppose the original message read as in line (1): (1) AAA CCC GGG TTT Now suppose also that one of the As is the nucleotide that is lost. Then the message will be read as in line (2): (2) AAC CCG GGT TT Then every codon from the break onward will be misread. The entire remainder of the gene, if a gene is involved, will be wrong, the resulting protein will probably be at least useless and possibly harmful. In cases of attempted replication, the result would almost certainly be lethal. Such an approach would be a disaster. There is an alternative. The system cannot know what to put in place of the missing nucleotide, but it dares not leave it blank or simply rejoin the parts. What it does, in fact, is to insert any two mutually complementary nucleotides, indiscriminately, one into each DNA strand. The two strands are thus both returned to their former complete size, intact, and still mutually complementary. There are four permanent nucleotides, so the chance that the repair will insert the right one is only 25%. In most cases, the choice will therefore not be the missing nucleotide. In such a case, an error will be present, but the system has no way to discover it. The result may make one enzyme defective, although not always. Usually one error will not be disastrous (we are full of DNA errors: a tiny proportion, but, because we have so many nucleotides, still a considerable number, yet usually not disastrous). Sometimes it will cause no noticeable problem. That is why mutations can survive, experience further mutations, and occasionally result in a useful advance. Indeed, a particular mutation will often be among nucleotides which are part of any gene, and usually these have no appreciable effect at all. The most important consideration, however, is that the reading will not be disrupted. The rest of the gene, if the problem was in a gene, remains correct (as correct as the template was), and the rest of the whole genome remains correct aside from the one wrong nucleotide in millions. It should be added that the system has no way to correct a missing block of nucleotides from both strands and will not do so. It cannot count how many are missing if its more than one. Perhaps an intelligent design might have allowed for the third case, a way to read past

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one at a time, or a way to read backward from a final stop code. But that is not how reality works. Altogether, these various refinements in the genetic system has increased the accuracy of replication and the making of proteins many times over, many orders of magnitude. Evolution, of course, did not stop with the protobions, even the most advanced ones. At least some bacteria and probably archaea as well have gone farther and the eukaryotes (including us) have progressed farther yet. Mutations and evolution continue, but for life to survive at all in its more complex forms, a very high accuracy and persistence rate are necessary. The systems for accomplishing these things (and others) are far more complete, complex, and fascinating than I have revealed here.

Step 55. DNA replication initiation.

One advantage of DNA over RNA is that DNA is more stable, even before it is paired with a second strand, surrounded by a protein capsid, and spared most exposure to stray molecules. It is therefore preferable to RNA as a permanent genome. It also has a greater tendency to remain flexible, not typically forming the special, fairly rigid, internal, matched stem, loop, and three-dimensional structures typical of RNA. This second characteristic can be helpful in fitting it into cramped spaces, generally necessary for a long genome, and thus opens the potential for very complex bions. On the other hand, it also means that DNA would not, and did not, initiate biology or operate as a single-molecule bion as RNA did, or convert parts of itself into ribozymes, stem-loop sensor-reactors, or create its own starter section, presumably a ribozyme originally, to initiate replication, as RNA also did. Then how does DNA replicate today, enabling all Kingdom 6 protobions and all cellular forms of biota (including us) reproduce? By using a special stretch of RNA as a kind of starter which chemists call a primer. Originally, a protobion (whose parent had been a passive bioid) created and became a member of the second kingdom by being, or having within itself, a section of RNA nucleotides that had a modest influence enabling it to initiate replication and designate the nucleotide (or base) at which to start the matching process. That set of adjacent nucleotides was the starter identified earlier, and was the first ribozyme. Undoubtedly, that ribozyme starter rapidly evolved to ever greater effectiveness, efficiency, and accuracy, within the potential of an RNA strand. When the genetic system developed far enough, enzymes were created which helped the reproductive process become more precise, more efficient, and more dependable. When the ssBRNA replication process later evolved to retain a complementary strand just made, it created Kingdom 3, the dsBRNA. In turn, the transitional Kingdom 4 consisted of RNA protobions matching their RNA with DNA nucleotides as the first step of a two-step replication process. The result necessarily was that the first DNA strand naturally formed as the chemical (electrical and spatial) complement to the RNA strand. In one sense, then, it contained complements to the RNA genes, but had no genes itself that would function as such without the reversing process. That situation was no problem. The advantage of this kind of arrangement was that the DNA, being a complement to the functioning RNA strand, could then assemble a whole series of functioning RNA strands, rapidly increasing its functioning descendants, without the waste of further non-functional RNA strands, which previously had necessarily occupied every second generation. Of course, the new system required new and improving or changing enzymes to make the whole thing work efficiently. When it reached a suitable point in that improvement, one Kingdom 4 bion discontinued the reproduction of complete, new RNA bions, instead copying

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parts, which shrank gradually until each part (in most cases) was a single gene, i.e., the set of nucleotides that code for one protein. This produced the double advantage of not having to produce so much RNA when only one protein was needed, of being able to produce the appropriate amount of each needed protein (and varying it as needed, regardless of what else might be needed at the same time). The discontinuance of reproduction of whole RNA protobions, however, could not help the Kingdom 4 transition group, or its descendants, unless the DNA could reproduce. As noted above, DNA does not, however, signal its own reproduction. It never has. The first DNA genome strands were, as previously observed, complements of functioning RNA strand (or in some cases of complements to functioning RNA strands). The complements of functioning strands, in those days, likely were largely non-functional by themselves. They were merely a record in DNA language of what the original RNA language instructions were. Thus, the DNA copy of a functioning RNA strand would complement all of it, including the ribozymes still there, such as the starter section. In DNA, that starter section complement does not start replication, however. When the DNA replicated numerous new RNA strands, it included that starter section. As that began to change to producing shorter RNA sections, one of those short bits of RNA was the starter section. When the system developed far enough to do so, the starter section was transcribed (as biochemists say) into an RNA starter, a descendant of the original one (much evolved), enzymes evolved to guide the parts of the developing new system, and a new DNA strand would be formed matching (complementing) each of the old ones. The new strand complementing one old strand (call it A) would then stay with A to make one dsDNA Kingdom 6 bion, and the new strand complementing the other old strand (call it B) would stay with B to make another bion, thus making two bions out of one. That has been going on ever since in all Kingdom 6 bions and all its more complex descendants. Of course, with this new reproductive system did not spring into being all at one time. Multiple gradual changes occurred in terms of mutations which produced small, gradual changes in enzymes, signal peptides and RNA stretches, amounts of mRNA formed, and bion behavior. In addition, while small changes in enzymes could enable the system to work using the old RNA starter to initiate a new DNA strand, no advantage remained to keeping the exact old form of the starter. It had originally been adapted to initiate RNA replication by its own chemical influence (ribozyme function). That influence could be gradually replaced by enzyme changes, perhaps leading to more efficiency and accuracy with DNA. At any rate, the present form of that ancient RNA starter for DNA reproduction, at least in us, is a relatively simple collection of sets of multiple successive copies of the nucleotides and is now called a primer. The new DNA strand is started by it in replication and attached to it. But the primer is removed when the new DNA strand is complete. As pointed out in relation to the previous step, once the sixth kingdom or protobiota was firmly in place and fully developed, new RNA strands were no longer initiated by the old starter. Rather, portions of them were produced as mRNA, usually as the original RNA gene for a particular protein (whether structural, enzymatic, or as a signal). Initiating the making of mRNA was done by activating a new kind of DNA section called a promoter for the particular mRNA needed. An enzyme then guided free-floating nucleotide triphosphates to the appropriate place for starting the building of the new strand. That remains true today in Kingdom 6 and in all cells including ours (except those in which, because of age in a particular case, have brought most internal processes to a stop, such as mature erythrocytes, i.e., red blood cells).

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Step 56. Sensors and ion catalysts.

We are aware now that protobions developed sensor-responders even in the ssBRNA stage, at first in the form of stem-loop structures within the main RNA strand. These were gradually refined, and, after the rise of the genetic system, began to acquire enzyme assistants or supplements. As time went by, shape changes and especially the special enzymes enabled the responding system to bring captured ions and molecules into contact with other structures which could use them. As the genetic system developed far enough to make molecules, whether enzymes or structures, refined enough to become very precise, specific, and effective in their functions, some of these molecules began to work together, sometimes joining themselves into strings or attaching the end of such protein strings to another part of the protobion. Later, such protein parts of the sensor-responder system seem to have taken over the entire function of that system. The former RNA parts then declined to vestigial organs, ceased to function, and disappeared altogether, especially after the dsRNA forms of Kingdom 6 developed fully. What would these sensor-responders collect? As we have seen, some of the early ones collected amino acids, and those evolved into tRNA molecules and perhaps the three main RNA molecules of the later ribosomes. Other likely collected other organic (carbon) compounds for use as parts of, or as materials to make parts of, new protobion offspring. Still others may have started collecting useful catalytic ions, mostly metals. We have noted that metal ions and metals in solids on the Ocean floor (or the midocean ridges of that floor) often served as catalysts for some early steps in building and maintaining protobions, such as the aluminum in montmorillonite clay, sulfur (a non-metal), metallic sulfides of copper, iron, calcium, magnesium, and the rest of the minerals necessary for biology mentioned in the partial atomic chart in step 3. Most of these were not directly necessary for early protobions but became increasingly important as bions ventured into more developed forms, usually one mineral for each new step. In the early days of that process, the collection may only have occurred at the time of immediate need, but reserve supplies may later have been built up for more regular use and more dependable reserve. The details of the more advanced steps in this process are reserved for later steps of this work. We have no direct evidence of exactly when this process began, but it must have been later than the steps that we have described, but earlier than the first successful cell. That limits us to approximately the last few tens of millions of years before 3.9 billion years ago, when cells first became numerous enough to affect the rocks forming on Earth.

Step 57. Another nucleotide.

Besides the five usual nucleotides that appear in nucleic acid strands (adenine, cytosine, guanine, and uracil in RNA, with the first three but thymine as the fourth in DNA), and the occasional modified nucleotides like inosine, there exists another kind, not previously mentioned here: nicotinamide adenine dinucleotide. This nucleotide does not become a part of a nucleic acid chain or strand, but has a different role in biology. The long name arises from two features of this nucleotide. First, it is composed of two different nucleotides bound to each other (this is what the di- in the last part of its name refers to). The adenine portion we have encountered before a purine with a large ring of six atoms, and a smaller ring of five atoms, these two rings sharing the two border atoms between them

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(on the lower right in the diagram). This purine consists of (1) the familiar heterocyclic organic base, with two nitrogen atoms in each ring, separated by one of the carbon atoms that make up the rest of the base rings; (2) the usual five-atom ribose ring, and (3) the usual phosphate ion (see accompanying figure).

Nicotinamide adenine dinucleotide (from Wikimedia Commons).

Second, the other part also has its own phosphate ion and ribose sugar ring, but the remaining ring, though resembling a six-atom, six-sided pyrimidine ring, has only one nitrogen atom within the ring (derived from nicotinic acid, at upper right of the diagram) and an attached side- or functional group called an amide. This ring, with its amide, is the nicotinamide of the name. The amide group consists, as shown, of a central carbon atom, attached or bound to the nicotinic acid ring with one of its four bonds, bound to an oxygen atom with a double bond, and to a nitrogen with the fourth bond (of the carbon atom). The amide nitrogen, in turn, is also bound to two different hydrogen atoms. This whole assemblage differs from nucleotides previously discussed (besides the amide side group and the single nitrogen in the ring instead of the two found in other nucleotide-base rings), in having its two nucleotides attached entirely between the two phosphate ions rather through a phosphate bond with the sugar of the other participating nucleotide, as is true in nucleic acid strands. Despite the oddities of this molecule, it seems universally to be present in all cellular biota, so it is likely to have become available to bions before the first cell arose. It plays a part in a large range of biochemical interactions involving removing or adding hydrogen atoms to a molecule. It is therefore not surprising that most biochemists avoid the long name and simply use its initials, NAD. NAD can accept hydrogen atoms from another molecule. In that early half-billion-year period of protobion development, hydrogen was plentiful, the Ocean was more acidic than now, molecules often needed to be changed by replacing a hydrogen atom, usually bonded to a

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carbon atom, with something else, often a functional group. The hydrogen ion would be attracted to NAD. When NAD has collected all the hydrogen atoms it can, it has become NADH2. It can also give up those two hydrogen atoms and be restored to NAD, usually then written NAD+2. As enzymes developed and became more precise and effective, NAD enabled them to remove hydrogen atoms from existing molecules (or it could provide them directly as NADH2). It thus became what we now call a co-enzyme, actually receiving the hydrogen atoms that were removed. An enzyme tends to be very large, surrounding all or part of the molecule that it is fitted to influence and, while it may change shape, it returns to its normal shape when the job is done. However, this co-enzyme is quite small compared to the usual enzyme (notwithstanding the multiple parts shown in the diagram) and is actually changed by its function, coming away from the interaction with a number of hydrogen atoms different from when it began. Only after the chemical interaction is completed can NADH2 return to its previous state, and even then only through other reactions. This new molecule and its functions will play a large part in the further chapters of this volume which will also touch on other enzymatic and co-enzyme interactions.

Step 58. Further co-enzymes.

Nicotinamide adenine dinucleotide (NAD), as outlined in the previous step, is a small molecule (compared to the long nucleic acid strands and proteins), a co-enzyme, in this case a hydrogen acceptor (or, NADH2, a donor) in the conversion of one molecule into another, which is a process catalyzed by an enzyme, normally a long protein. Other co-enzymes also exist, so called because today they generally play a vital part in a process usually catalyzed by an enzyme. Their origin is not certain but likely was relatively ancient, in this case almost certainly before cellular bions came into existence. Today they are built up by biological actions, that is to say, bions build them. (a) Coenzymes or something like them may have first arisen by non-biological processes and came to be used by bions, perhaps even before protobions could create peptides or proteins. The method of use may even have involved attachment to an RNA strand, but that is not proven by known examples. At a later time, the bions that used them may have adapted to a need for readier access by making occasional single-atom changes to very similar molecules to convert them into those early, pre- or proto-co-enzymes or something closely resembling them and able to perform an approximation of their functions. In such a case, further mutations would have led to changes of a second atom for greater refinement or enabling a two-step conversion of a molecule one step farther from the needed co-enzyme, and so on repeatedly until the entire co-enzyme could be made through a number of steps from one or more molecules commonly available. So, co-enzymes often resemble other molecules, including RNA nucleotides and each other. The origin suggested under (a) in the two paragraphs above seems most likely to me but is not proven by examples of interim steps existing in separate bions. (This kind of origin is, however, is well known for many molecules, though not specifically for this one.) Alternatively, probably less likely, is (b) the independent origin of a series of mutations leading to each new co-enzyme, or (later) (c) adaptation of one existing co-enzyme or other bion product into a new, needed co-enzyme. Generally, neither (a) nor (b) is definitely proven, while (c) seems surely to have occurred in at least one case and probably others. That one case is the rise of NADP, which is exactly the same as NAD except than an extra phosphate ion (or radical) is

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bound to the lower, right-hand oxygen atom attached to the right-hand pentose sugar, as shown in the diagram above, in place of the hydrogen atom that was there in simple NAD as shown. NADP (nicotinamide adenine dinucleotide phosphate) performs the same function as simple NAD, though often one of these two co-enzymes is typically used (preferred) or more efficient in one such reaction than the other. Adding another phosphate ion (or losing it) in the early biological environment was such an easy and simple step that one of these two coenzymes must surely have arisen from the other and both were used interchangeably. (When a NADP molecule has accepted two hydrogen atoms and is then able to act as hydrogen donor, the two extra hydrogen atoms are shown as NADPH2). Many other co-enzymes exist, including: (n) flavin mononucleotide (FMN), also known as riboflavin (vitamin B2) phosphate, which helps move hydrogen atoms from NAD or NADP to cytochromes (a class of proteins containing a metal ion catalyst); (o) flavin adenine dinucleotide (FAD) with a similar function; (p) co-enzyme A (co-A); (q) co-enzyme Q; etc. A number of enzymes and co-enzymes (not just the cytochromes) contain metal-ion catalysts, as outlined in the next step. Those whose structural formulas I have seen look as though they may have been derived from each other, but I am not aware of any specific history confirming that. Some co-enzymes look as though they probably were originally used before the relevant enzymes arose. (The reader may have noticed that some of these co-enzymes have names implying that they are, or are composed of, nucleotides, and that term is used for them, because they are indeed regarded as nucleotides because of similarity of shape, structure, and composition. Like RNA and DNA nucleotides, they each contain phosphate ions, pentose sugar, and a third element, and this third element may resemble a purine or pyrimidine base in general shape. Yet today they are unknown as parts of RNA or DNA strands. It is conceivable that they once were members of or attached to RNA strands, or even that they were first made of one or more RNA nucleotides in a strand of RNA. That would be plausible but not necessarily more so than that several similar types of molecules first became available through non-biological processes.)

Step 59. Metal-ion catalysts, pyrroles, and porphyrins.

Neither sensor-reactors, aside from their role as the origins of tRNA molecules, nor coenzymes, nor most enzymes, nor metal-ion catalysts, nor pyrroles, nor porphyrins are parts of the Kingdom 6 genetic system in the sense of being DNA gene coders or the various kinds of RNA decoders or assemblers of amino acids to build proteins. Many enzymes are not involved in those processes other than those specifically functioning to collect the amino acids, attach them to the pertinent RNA molecules, or to bind them together. Even so, the items touched on in steps 56-59 are all (except the original sensor-reactors and the metal ions) the products of the genetic system and so may generally be described as among the effectuators of the ultimate functions which that system made possible. These are all parts of all bions more complex than the protobion kingdoms 1-5, and are prerequisites to acquiring the remainder of the widespread characteristics of biology listed earlier. That is why they are included in this chapter. As we are aware from step 3 and its accompanying chart, numerous elements are crucial to biology besides hydrogen, carbon, nitrogen, oxygen, sulfur, and phosphate, which

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have dominated our history up to this point. It is especially the metal ions that are crucial. Even today they remain absolutely necessary for many functions in bions and, at the earliest phases of biology, and some phases of necessary pre-biology, they played an even larger part. Yet none of the early protobions, nor even those existing today, have been proven to actually contain (or to have contained at any time) most of those metal ions within their own structure. Even so, early bions could take advantage of the processes that those elements enhanced and even come in contact with them in solution or on the Ocean floor. Even the viruses of today can survive without known direct use of such catalysts because they reproduce mainly in other bions (cells) which they parasitize. But as those ancient, Kingdom 6 dsDNA protobions moved toward more complex lives, the use of metal-ion catalysts became necessary for the next steps toward efficiency and control. Before considering how that happened, let us examine the following list, contained in one of our written sources (see bibliography after chapter N).55
55

The author may refer here to Wagner, E.K., M.J. Hewlett, D.C. Bloom, and D. Camerini. 2007. Basic Virology. Hoboken, NJ: Wiley-Blackwell. PowerPoint slides and images from this book are available online: www.blackwellpublishing.com/wagner/artwork.asp. For further information on the origin of coenzymes, see Chen, X., N. Li, and A.D. Ellington. 2007. Ribozyme catalysis of met abolism in the RNA, in Chemistry & Biodiversity 4 (4): 633-655. Doi:10.1002/cbdv.200790055; Ouzounis, C. and N. Kyrpides. 1996. The emergence of major cellular processes in evolution, in Federation of Biochemical Letters 390 (2): 119-123. Doi:10.1016/0014-5793(96)00631-X; Koch, A. 1998. How did bacteria come to be? in Advances in Microbial Physiology 40: 353-399. Doi:10.1016/S0065-2911(08)60135-6; White, H.B. 1976. Coenzymes as fossils of an earlier metabolic state, in Journal of Molecular Evolution 7 (2): 101-104. Doi:10.1007/BF01732468; Saran, D., J. Frank, and D.H. Burke. 2003. The tyranny of adenosine recognition among RNA aptamers to coenzyme A, in BMC Evolutionary Biology 3: 26. Doi:10.1186/14712148-3-26.

Range of the percentages of body weight of the main atoms in cellular life on Earth Primary Secondary Minute 1-60% 0.05-1% 0.05% or less C H N O P Na Mg S Cl K Ca B Fe Si Mn Cu I Co Mo Zn

As protobions became more numerous and perhaps even more competitive, and as the most advanced of them entered the phase described in the next chapter and beyond, one limiting factor in their lives became dependence on chance to take them to the appropriate metallic catalysts at the right times and in the right circumstances (or to bring such substances to them). One imaginable way to deal with such a problem is to collect and save such ions for times when circumstances suggest their use. Conceivably this could be done in either of two ways: a sensor-reactor could recognize and seize them, or a protein perhaps an enzyme could collect and usher them to the point where they might be needed.

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Both ways present difficulties. The permanent DNA genome had taken the place of the permanent BRNA molecule and, by this point, RNA had come to be manufactured only for shortterm purposes, after which the RNA was generally recycled, unless it was too specialized to be useful for this new role. So how would the sensor-reactor remain tethered to the bion? We do not know the answer to that, although it must have been achieved in some fashion. Perhaps the association was with the capsid, or some part of it, or with a surface to which the bion adhered. Beyond that stood the problem of retaining a metallic ion in a way that kept it usable. Either some sort of sensor-reactor, whether composed of RNA or protein, or an enzyme or other protein, might react with a metallic ion by forming a bond. But after that such an ion on the end of a strand of RNA or protein might be attacked by another atom or molecule. For example, the Ocean contains abundant salts. The most common salt is composed of sodium (Na) and chlorine (Cl). Both of those ions are highly reactive. They normally attract each other more than either is attracted to carbon in the bion. But when metallic ion is attached to a strand of RNA or protein, a salt molecule may react to that metallic ion, in effect, removing it. Thus, even if a metallic ion is captured, it may easily be stolen away by other passing ions and molecules. Further, different atoms have different degrees of chemical (electrical) attraction to each other and carbon is well down the list. The consequence is that if the bion, or a particular product of it that needs to bind with a particular kind of ion, say, calcium, magnesium, or manganese, does so, another metal ion with a higher binding power or attraction, say sodium or potassium (K), will replace it, preventing the bion from retaining what it needs. Finally, even if those problems are overcome, the binding may either be so strong that the ion cannot be used to do its catalytic job, or it may attract some other atom or molecule to it in a way that interferes with the needs of the bion. It turns out that this problem was solved by developing a special kind of carbon structure, a kind of prosthetic group, as it is sometimes called. Typically, an enzyme is made entirely of protein (a long strand of many specific amino acids, arranged in a specific order). Some enzymes, however, need a relatively small nonprotein part. That is the prosthetic group. (In other contexts, prosthetic means helping. In medicine, for example, a prosthetic device is some non-living mechanism which may be brought into, strapped onto, or carried by the patient, either to pump some needed medication in, to induce the heart to follow a set pace of beating, to reopen or replace a tube needed for moving blood or other crucial fluid, a hearing aid, a cane, crutch, or eyeglasses, replace a damaged or removed bone or joint, etc.). In this case, the prosthetic is merely the non-protein part of the enzyme, helping the enzyme by doing what the protein part (the apo-enzyme) cannot complete by itself. For collecting, retaining, and making available metal ion catalysts, the prosthetic group is built of four five-sided, hetero-ring pyrroles. A pyrrole molecule has roughly the shape depicted in the figure below:

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Pyrrole (from Wikimedia Commons), four of which comprise a prosthetic group.

Note that this pyrrole is a five-atom ring, unlike the six-atom pyrimidine ring, and has one nitrogen atom (N) as part of the ring itself, rather than the two nitrogen atoms in the purine and pyrimidine rings. The genetic system, through several chemical steps mediated by several appropriate enzymes, build a flat but otherwise symmetrical structure by hooking four of these pyrrole molecules together into a larger ring or box with individual carbon atoms each linking two pyrroles to each other, at right angles in the same plane. The nitrogen atom in each pyrrole points toward the center of the large ring, as shown in the accompanying diagram.

Structure of porphine, the simplest porphyrin, showing the larger ring or box structure, with carbon atoms at each unmarked angle (from Wikimedia Commons, article porphyrin).

This structure is called a porphyrin structure or ring and is a prosthetic group. (Other kinds of prosthetic groups also exist for other purposes.) The outside edges of this structure are bonded in the enzyme, keeping the whole porphyrin rings in this flat shape. This porphyrin ring is a place for collecting, keeping, and using a metal ion (with two valence electrons) as a catalyst in a chemical interaction. The divalent metal ion, such as calcium (Ca), magnesium (Mg), manganese (Mn), copper (Cu), iron (Fe), etc., is caught and kept in the very center of the porphyrin structure, single-bonding with each of two nitrogen atoms on opposite sides of it. In this way, the metal-ion catalyst uses both its available bonds, holding it in place and reducing the likelihood of its being stolen away by some passing atom or molecule. That likelihood is further reduced by the ions nearness to the other two nitrogen atoms as well, and by the precise and rigid placement of those nitrogen atoms all the way around the ion in one plane.

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Heme B group of hemoglobin, showing an iron (Fe) atom in the center in red, complexed to four interior nitrogen atoms in blue, an example of a porphyrin holding a metal ion in place (from Wikimedia Commons, article porphyrin).

Yet at the same time, the enzyme can fold and position itself in such a way that the whole flat structure can be brought close to the crucial site where prospective chemical interaction is needed, because the flat side of the porphyrin can be brought right against that site. The ion is thus brought exactly to where it is needed without much risk of its loss before it gets there. When the porphyrin is intact and contains the metal ion, it has the effect of producing an opaque color, making a biological pigment. Different kinds of ions yield different colors. This kind of structure is ordinarily used for ion with two valence electrons. All of the nitrogens help to attract the metal ion as well as to hold it in a rigid position. Their combined negative electric charges tend to repel any negatively charged passing atoms or molecules, protecting the catalyst from theft, while passing atoms or molecules with positive charges will not attract the positively charged metal ion. We do not know exactly when this system was developed or through how many intermediate stages it may have gone, to reach this find adaptation to its function, but it must have been after protobion Kingdom 6 arose and before cells did, so it must be close to 3.9 or 4 billion years ago, when the cells arose. All two-valence metal ion catalysts used by biota in enzymes seem to be held in this kind of structure, so it has been a great success, used by all cellular life since it arose (as far as has been discovered). It has stayed the same since reaching that refined but still relatively simple state. (Making it from four similar parts, adaptable to any divalent metal, helps make its production relatively simple and efficient, provided the elements are available. It is usable for any divalent metal ion, but the pertinent enzyme can sense whether it contains the correct metal.) Almost all metal ions used by biota are, in fact, used for their catalytic influences. Exceptions are calcium carbonate, found in shells, teeth, and bones, and iron which attracts and holds oxygen, giving us our red blood and pinkish skin (if we do not have enough skin pigment to hide this pink). After refinement of this structure and its use, biota have not had to wait so

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much or so helplessly until finding needed metals (commonly calls minerals by dieticians), unless living in areas of actual shortages of certain metals. (Such areas do exist. Trade can reduce the problems that they create but has not done so entirely.) (Side note: when dietary supplements are called chelated minerals, they refer to metal ions held in such a porphyrin structure.)

Step 60. Protobions in the larger chemical Ocean.

In this chapter, we have watched the progress of the former protobiota as they originally existed, consisting primarily and almost entirely of nucleic acid, until they developed to a much more complex and complete system with dsDNA, with various specialized ssRNA molecules, large and complex proteins, co-enzymes, and prosthetic groups, particularly metalion catalysts in pigmented porphyrin structures. We have also mentioned the six most prominent elements or atoms in this process: carbon, hydrogen, oxygen, and nitrogen from the air of the early Earth, caught in water, and in the Ocean brought, to some degree, into the areas of the deep-Ocean ridge system, where they are joined especially by phosphorus and also, though not emphasized yet, sulfur (often summarized by their acronymic symbols, CHONPS). Besides these, many metal-ion catalysts played their parts, not fully described so far, but finally resulting in their incorporation with the development of the porphyrin rings to hold them. We might also note that the six atom types above (except hydrogen) are the main atoms within biology that multiple bonds, i.e., bonds with more than one other atom at a time. Beyond the protobiota themselves, and apart from their micro-sites, where they likely attached themselves and their products to various particle surfaces by electrical attractions, or to each other, they were also immersed in, and in a sense constituted parts of, the Ocean. That Ocean was composed of water, HOH (or H2O), which, in turn, consisted, as it does today, of two associated but separable ions, a positively charge hydrogen ion (H+) and a negatively charged hydroxyl ion (OH-). If these two ions are present in equal numbers, the result is a neutral Ocean. If an excess of H+ (hydrogen ions) is present, then the Ocean is acidic. If an excess of OH- is present, then the Ocean is basic. This balance, and its degrees of tilting one way or the other, affect chemical transactions within it. In fact, the ancient Ocean of the time covered by this volume the first half a billion years of pre-biological, chemical and pre-cellular, biological or biochemical evolution was somewhat acidic, in contrast to todays Ocean. That made many chemical interactions more (and some less) likely or efficient than today. Likewise, every other set of chemical relationships also affects a balance in the Ocean, just as the balance of H+ versus OH- does. The relative proportions of every kind of available ion affected interactions of every other ion present (as it does now). The proportion of salt affects the saltiness, the proportion of hydrogen ions affects the sourness, etc. (We have words like saltiness and sourness for some chemical effects that we consciously taste, but a myriad of other chemical phenomena affect biology in ways for which we have no common words, because our conscious senses do not directly detect them.) As often in natural systems, each element affects all, in a complicated fashion. Some of these are more important to biology than others, and the whole of biology, while important to those bions that are parts of it (like us), is slight compared to the whole Earth, or even to its crust or the Ocean. Still, biology has consistently been more extensive and a larger part of the Ocean than of the crust of the Earth.

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Our ancient, cell-less ancestors shared the Ocean with innumerable other molecules, forming, being modified, being broken, and changing in various ways dependent on the properties of the atoms of which they were composed, and to which they were exposed. At the time, a sentient being likely would not have noticed the earliest bions as anything distinctive from all the other molecules and processes of the time, and might not have been able to foresee their impressive future, even if equipped with proper tools to permit recognizing them. They were embedded, chemically speaking, in a sea of interactions, driven by energy sources, but in most cases directionless. Without a genetic system, the various miscellaneous interactions of whatever sort that could interact likely led to cycles of repetitive, sequential interactions, and all interactions probably rapidly developed into a web of related interactions. Yet without a genetic system to guide and preserve structures and functions useful for the survival of the genetic system, this background chemistry would be unable to evolve beyond a certain set of interrelated cycles. Yet that separate, broader set of interrelated events, not yet constituting any real function without the driving force of even natural selection, was available for bions to being to utilize, if that would assist them. That is increasingly what happens during the remainder of this book. We might note, too, that among the four pertinent types of atmospheric atoms on the early Earth, the different strengths of chemical bonds affected (and continues to affect) the functional relationships among them. Here are some examples:
This first column of bonds are all low-energy bonds; hence sites of chain cutting or termination. The second column consists of medium-energy bonds, so used for extending chains. The four bonds on the far right have high energy and so are used for the ends of chains, because they can hold firmly and will not be taken away by passing atoms or molecules. The double bond between two oxygen atoms, shown as O=O, is a relatively high-energy, chain-terminating bond.

H-O H-H H-C H-N C=O NN N=N NC

C-O C-C N=C C=C N-C CC N-O N=N N-N O-O

HONC-

The special advantages of phosphorus, and in particular of phosphate ions, the form of phosphate normally used in biology, are that (1) phosphorus also can form multiple bonds with different atoms, (2) phosphate ions form bonds rather easily, though it is high-energy bond, easily added from pyrophosphate, and (3) the bonds can also easily be broken, providing chemical energy, which can be coupled to a synthesis of a growing molecule, enabling biology to begin and to grow ever bigger molecules with wider functions. As mentioned, phosphates provided the original, main, chemical energy source for late pre-biological and many later biological reactions. Phosphates were mainly available along the deep-Ocean ridges constantly built by geothermal vents and plumes, to begin with. And they remain the main energy source

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for most processes inside cells, even far from that original source, but derived more indirectly and in a more sophisticated way from other chemical reactions. One author emphasizes and deals with the important issue of the origin and interrelationships of the many parts of that general category of biochemical processes involving (1) collection of matter to be used later, (2) the building of molecules, (3) the destruction of molecules, (4) the storage of molecules, (5) their use in making other structures and producing energy, (6) the use of that energy, and (7) the discard of unusable matter. These processes, when collected together and interrelated, are called metabolism. Rejecting the idea of life before its cellular form, on the basis that it could not be distinguished from the sea around it, this author still recognizes that even the simplest cell is far too complex to spring suddenly into being, notes that much of (chemical potential) energy storage consists of molecules lacking nitrogen, and thus arrives at the view that the earliest evolution toward biology arose from an evolution of that general system of chemical interactions of the Ocean relating some hydrogen, carbon, and oxygen compounds. He even adopts a slogan or aphorism to represent his idea: Metabolism recapitulates biogenesis.56
56

Morowitz, Harold J. 1992. Beginnings of Cellular Life: Metabolism Recapitulates Biogenesis. New Haven, Connecticut: Yale University Press.

His 1990s slogan appears to be inspired by a 1920s slogan ontogeny recapitulates phylogeny.57 His idea is that relationships he sees among metabolic molecules (metabolism) must show the way to the origin of biology (biogenesis). The 1920s slogan meant that stages in the maturing of an individual (ontogeny) repeat the adult stages of some of the individuals ancestors (phylogeny). Interestingly, both slogans, and the ideas they represent, are mistaken as stated and meant, but on deeper analysis can be seen to hold a germ of truth, just like some of the examples in chapter B showed some inaccurate ideas still suggestive of an actual part of the answer, like the mud-genes idea, clearly wrong, but relevant to the importance of metal-ion catalysts. (Indeed, this is often one way in which science can progress. A wrong idea, if popular enough, will likely be tested. The results will often reveal the error and may lead to discovering a misunderstood aspect of the reality.)
57

The first formal statement of this concept was by tienne Serres in 1824-26 as what came to be known as the Meckel-Serres Law, according to Wikipedia.

In the 1920s case, the attractive sounding (though obscurely stated) phrase was not correct as stated. But there is some tendency for complex bions going through a long series of changes to adulthood often to pass through, as embryos, forms quite similar to the embryonic forms of some of their ancestors, as we shall see in later volumes of this series. (This should not be surprising and illustrates the incremental nature of evolution as opposed to the periodic misreadings that lead to assertion of jumping from one form to a strikingly different one. Those cannot happen; they appear to only because the intermediate forms are not preserved the odds against survival of any fossils are astronomical, except for extremely large, bony forms, and only a very tiny fraction do survive.) Likewise, in the present case, the lack of genetic system to preserve useful and discard useless or harmful aspects in these non-biological forms made any real evolution impossible, not only for lack of those control functions, but also for lack of any mechanism or cause for distinguishing between useful and harmful. Where there is no genetic system to preserve and strengthen, there is no entity to which usefulness or harm can pertain. But there remains this

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consideration: a web of chemical changes was happening in the Ocean, creating a set of cycles because these interactions could get nowhere outside their existing potential, and some of those may well have underlain some kinds of molecules that later became parts of metabolism, under the direction of nucleic acid genetic control. That is where the remaining chapters are headed, in part, and that aspect of genetic refinement led to a new form of bion, the prokaryote cell. Before proceeding to the chapters touching on those steps, however, we should consider one more step in the fundamental characteristics of protobions relevant to their genetic system, a step still detectible in our time and in existing bions. We have met the early, relatively simple capsids protecting the basic nucleic-acid strand of the bion. These were built of proteins, after refinement of the genetic system proper became able to construct them. The parts were so structured as to come together by electro-chemical attraction and because of their flat, multi-sided shapes, so they fit together into a hollow, icosahedral form (in the simplest form, having 20 identical facets, attached together in this shape by the angles among the directional relationships of electrical bonds of the component atoms along the edges of the plates). Others became more complex, with more layers in the capsid, and sometimes variation of composition among the layers of one bion. In one case, as the reader will recall, a protobion whose nucleic-acid strand was separated into multiple fragments, each enclosed in separate but similar capsids, except that they were each a different size, so that all capsids nested together, one inside another. That model proved to be a dead end. With dsDNA, the separate mRNA model, and increasingly complex capsids, broken strands no longer played major part of the more complex bions (though they still exist in something like their ancient form). All of these examples have already crossed our path, up to and including the still more complex capsids, with multiple layers to protect the DNA strands, each made of different material performing a different, specialized function, and containing additional inclusions, such as already-made protein enzymes and supportive structural proteins. Yet, consider one more incremental step, although, of course, like most steps mentioned up till now, it really consisted of many slight mutations over substantial time. This was a further refinement, leading (a) to more inclusions within the capsid, such as catalytic metal ions embedded in porphyrin-ring structures, perhaps more enzymes, better protein support structures for the DNA, and perhaps even some items from the next chapter. Besides all this, sometimes else occurred, actually still existing in a few instances: (b) better, larger, more complex, and more sophisticated capsids themselves. What appear to be some additional support structures for the capsids themselves, and additional layers often characterized as part of linings of other capsids, but constituting still further layers, appeared. Some became large enough that the angular, faceted appearance, while often remaining for an inner capsid, seems to disappear in favor of more rounded-looking, outermost capsids. At this level, though barely detectible with the most powerful microscopes of today, these have reached a size and complexity that hides any angular components in the overall outside shape, a characteristic common to most larger and more familiar bions and their parts of larger size. It beings to look like a more familiar, visible bion, without sharp angles in general shape, becoming more rounded, though often longer than wide, with an increasingly complicated internal structure.58 (Note: for simplicity, consistency, and avoidance of the several terms that specialists use for various layers of capsids of protobiota, only the word capsid is used for all of them here. Most specialists use that word only for the innermost enclosure of the nucleic acid, and use other terms to identify the other layers. Those outer structures have not been described as

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having any successors surviving in us, so their details, though reported, have not been covered here.)
58

The mimivirus is the largest known, a genus containing at least two individuals, Acanthamoeba polyphaga mimivirus and Megavirus chilensis. The term mimivirus is an abbreviation of the phrase mimicking microbe, which reflects its large size and the fact that it is so big it was at first mistaken for a bacterium. The first of these giant viruses has a capsid diameter of 400 nanometers, with filaments of protein projecting from the capsids surface bringing the total length of this bion to 600 nanometers. Its genome is dsDNA with 1,181,404 base pairs, which is larger than at least 30 cellular clades. It also has 979 protein-coding genes, far more the minimal four required (the minimum is found in MS2 and Q viruses). These facts appear in Wikipedia, article mimivirus, citing, among others, La Scola, B., S. Audic, C. Robert, L. Jungang, X. de Lamballerie, M. Drancourt, R. Birtles, J.M. Claverie, and D. Raoult. 2003. A giant virus in amoebae, in Science 299 (5615): 2033. Doi: 10.1126/science.1081867; Xiao, C., Y.G. Kuznetsov, S. Sun, S.L. Hafenstein, V.A. Kostyuchenko, P.R. Chipman, M. Suzan-Monti, D. Raoult, A. McPherson, and M.G. Rossmann. 2009. Structural Studies of the Giant Mimivirus, in PLOS Biology 7 (4): 392. Doi: 10.1371/journal.pbio.1000092; Suzan, Monti, M., B. La Scola, and D. Raoult. 2006. Genomic and evolutionary aspects of mimivirus, in Virus Research 117 (1): 145-166; and Prescott, Lansing M. 1993. Microbiology. Dubuque, IA: William C. Brown Publishers.

An image of Megavirus chilensis, the largest known virus (from Wikipedia Commons, article Megavirus).