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PROTEIN SYNTHESIS

Table of Contents
One-gene-one-protein | The structure of hemoglobin | Viruses contain DNA
RNA links the information in DNA to the sequence of amino acids in protein
Transcription: making an RNA copy of a DNA sequence | The Genetic Code
Protein Synthesis | Mutations redefined | Links

One-gene-one-protein | Back to Top


During the 1930s, despite great advances, geneticists had several frustrating
questions yet to answer:
What exactly are genes?
How do they work?
What produces the unique phenotype associated with a specific allele?
Answers from physics, chemistry, and the study of infectious disease gave rise
to the field of molecular biology. Biochemical reactions are controlled by
enzymes, and often are organized into chains of reactions known as metabolic
pathways. Loss of activity in a single enzyme can inactivate an entire
pathway.
Archibald Garrod, in 1902, first proposed the relationship through his study of
alkaptonuria and its association with large quantities "alkapton". He reasoned
unaffected individuals metabolized "alkapton" (now called homogentistic
acid) to other products so it would not buildup in the urine. Garrod suspected
a blockage of the pathway to break this chemical down, and proposed that
condition as "an inborn error of metabolism". He also discovered alkaptonuria
was inherited as a recessive Mendelian trait.
George Beadle and Edward Tatum during the late 1930s and early 1940s
established the connection Garrod suspected between genes and metabolism.
They used X rays to cause mutations in strains of the mold Neurospora. These
mutations affected a single genes and single enzymes in specific metabolic
pathways. Beadle and Tatum proposed the "one gene one enzyme hypothesis"
for which they won the Nobel Prize in 1958.

Since the chemical reactions occurring in the body are mediated by enzymes,
and since enzymes are proteins and thus heritable traits, there must be a
relationship between the gene and proteins. George Beadle, during the 1940s,
proposed that mutant eye colors in Drosophila was caused by a change in one
protein in a biosynthetic pathway.
In 1941 Beadle and coworker Edward L. Tatum decided to examine step by
step the chemical reactions in a pathway. They used Neurospora crassa as an
experimental organism. It had a short life-cycle and was easily grown. Since it
is haploid for much of its life cycle, mutations would be immediately
expressed. The meiotic products could be easily inspected. Chromosome
mapping studies on the organism facilitated their work. Neurospora can be
grown on a minimal medium, and it's nutrition could be studied by its ability
to metabolize sugars and other chemicals the scientist could add or delete
from the mixture of the medium. It was able to synthesize all of the amino
acids and other chemicals needed for it to grow, thus mutants in synthetic
pathways would easily show up. X-rays induced mutations in Neurospora,
and the mutated spores were placed on growth media enriched with all
essential amino acids. Crossing the mutated fungi with non-mutated forms
produced spores which were then grown on media supplying only one of the
20 essential amino acids. If a spore lacked the ability to synthesize a particular
amino acid, such as Pro (proline), it would only grow if the Proline was in the
growth medium. Biosynthesis of amino acids (the building blocks of proteins)
is a complex process with many chemical reactions mediated by enzymes,
which if mutated would shut down the pathway, resulting in no-growth.
Beadle and Tatum proposed the "one gene one enzyme" theory. One gene
codes for the production of one protein. "One gene one enzyme" has since
been modified to "one gene one polypeptide" since many proteins (such as
hemoglobin) are made of more than one polypeptide.

The Beadle and Tatum experiment that suggested the one gene one enzyme hypothesis.
Images from Purves et al., Life: The Science of Biology, 4th Edition, by Sinauer
Associates (www.sinauer.com) and WH Freeman (www.whfreeman.com), used with
permission.

The Structure of Hemoglobin | Back to Top


Linus Pauling used electrophoresis to separate hemoglobin molecules. Sicklecell anemia (h) is a recessive allele in which a defective hemoglobin is made,
ultimately causing pain and death to those individuals homozygous recessive
for the trait. Pauling reasoned that if Beadle and Tatum were correct, there
should be a slight (but detectable) difference between the structure of a normal
(HH) and sickle cell (hh) hemoglobin due to genetic differences.
Heterozygotes (Hh, also sampled by Pauling) make both normal and "sickle
cell" hemoglobins. Later, Vernon Ingram discovered that the normal and
sickle-cell hemoglobins differ by only 1 (out of a total of 300) amino acids.
Viruses Contain DNA | Back to Top
The coats of viruses act as antigens, initiating an antigen-specific antibody
response. Remember that vaccines work by either prompting the immune
system to make antibodies or by supplying antibodies. If a virus (or anything
else for that matter) mutates its antigens, the immune system is forever
playing catch-up.
RNA Links the Information in DNA to the Sequence of Amino Acids in
Protein | Back to Top

Ribonucleic acid (RNA) was discovered after DNA. DNA, with exceptions in
chloroplasts and mitochondria, is restricted to the nucleus (in eukaryotes, the
nucleoid region in prokaryotes). RNA occurs in the nucleus as well as in the
cytoplasm (also remember that it occurs as part of the ribosomes that line the
rough endoplasmic reticulum).
Scientists for some time had suspected such a link between DNA and proteins.
Cells of developing embryos contain high levels of RNA. Rapidly growing E.
coli has half its mass as ribosomes. Ribosomes are 2/3 RNA (a type of RNA
known as ribosomal RNA or rRNA) and 1/3 protein. RNA is synthesized from
viral DNA in an infected cell before protein synthesis begins. Some viruses,
for example Tobacco Mosaic Virus (TMV) have RNA in place of DNA. If
RNA extracted from a virus was injected into a host cell the cell began to
make new viruses. Clearly RNA was involved in protein synthesis.
Crick's central dogma. Information flow (with the exception of reverse
transcription) is from DNA to RNA via the process of transcription, and
thence to protein via translation. Transcription is the making of an RNA
molecule off a DNA template. Translation is the construction of an amino acid
sequence (polypeptide) from an RNA molecule. Although originally called
dogma, this idea has been tested repeatedly with almost no exceptions to the
rule being found (save retroviruses).

The central dogma. Image from Purves et al., Life: The Science of Biology, 4th
Edition, by Sinauer Associates (www.sinauer.com) and WH Freeman
(www.whfreeman.com), used with permission.

The blue-background graphics throughout this chapter are from the University
of Illinois' DNA and Protein Synthesis site.

Messenger RNA (mRNA) is the blueprint for construction of a protein.


Ribosomal RNA (rRNA) is the construction site where the protein is made.
Transfer RNA (tRNA) is the truck delivering the proper amino acid to the site
at the right time.

RNA has ribose sugar instead of deoxyribose sugar. The base uracil (U)
replaces thymine (T) in RNA. Most RNA is single stranded, although tRNA
will form a "cloverleaf" structure due to complementary base pairing.
Transcription: making an RNA copy of a DNA sequence | Back to Top
RNA polymerase opens the part of the DNA to be transcribed. Only one strand
of DNA (the template strand) is transcribed. RNA nucleotides are available in

the region of the chromatin (this process only occurs during Interphase) and
are linked together similar to the DNA process.

Transcription of a segment of DNA to form a molecule of RNA. The above


images are from http://www.biosci.uga.edu/almanac/bio_103/notes/may_23.html.

The Genetic Code: Translation of RNA code into protein | Back to Top
The code consists of at least three bases, according to astronomer George
Gamow. To code for the 20 essential amino acids a genetic code must consist
of at least a 3-base set (triplet) of the 4 bases. If one considers the possibilities
of arranging four things 3 at a time (4X4X4), we get 64 possible code words,
or codons (a 3-base sequence on the mRNA that codes for either a specific
amino acid or a control word).
The genetic code was broken by Marshall Nirenberg and Heinrich Matthaei, a
decade after Watson and Crick's work. Nirenberg discovered that RNA,
regardless of its source organism, could initiate protein synthesis when
combined with contents of broken E. coli cells. By adding poly-U to each of
20 test-tubes (each tube having a different "tagged" amino acid) Nirenberg

and Matthaei were able to determine that the codon UUU (the only one in
poly-U) coded for the amino acid phenylalanine.

Steps in breaking the genetic code: the deciphering of a poly-U mRNA. Image
from Purves et al., Life: The Science of Biology, 4th Edition, by Sinauer Associates
(www.sinauer.com) and WH Freeman (www.whfreeman.com), used with permission.

Likewise, an artificial mRNA consisting of alternating A and C bases would


code for alternating amino acids histidine and threonine. Gradually, a
complete listing of the genetic code codons was developed.

Deciphering the code: poly CA. Image from Purves et al., Life: The Science of
Biology, 4th Edition, by Sinauer Associates (www.sinauer.com) and WH Freeman
(www.whfreeman.com), used with permission.

The genetic code consists of 61 amino-acid coding codons and three


termination codons, which stop the process of translation. The genetic code is
thus redundant (degenerate in the sense of having multiple states amounting to
the same thing), with, for example, glycine coded for by GGU, GGC, GGA,
and GGG codons. If a codon is mutated, say from GGU to CGU, is the same
amino acid specified?

The genetic code. Image from Purves et al., Life: The Science of Biology, 4th
Edition, by Sinauer Associates (www.sinauer.com) and WH Freeman
(www.whfreeman.com), used with permission.

Protein Synthesis | Back to Top


Prokaryotic gene regulation differs from eukaryotic regulation, but since
prokaryotes are much easier to work with, we focus on prokaryotes at this
point. Promoters are sequences of DNA that are the start signals for the
transcription of mRNA. Terminators are the stop signals. mRNA molecules
are long (500- 10,000 nucleotides).
Ribosomes are the organelle (in all cells) where proteins are synthesized. They
consist of two-thirds rRNA and one-third protein. Ribosomes consist of a
small (in E. coli , 30S) and larger (50S) subunits. The length of rRNA differs

in each. The 30S unit has 16S rRNA and 21 different proteins. The 50S
subunit consists of 5S and 23S rRNA and 34 different proteins. The smaller
subunit has a binding site for the mRNA. The larger subunit has two binding
sites for tRNA.

Subunits of a ribosome. Image from Purves et al., Life: The Science of Biology, 4th
Edition, by Sinauer Associates (www.sinauer.com) and WH Freeman
(www.whfreeman.com), used with permission.

Transfer RNA (tRNA) is basically cloverleaf-shaped. tRNA carries the proper


amino acid to the ribosome when the codons call for them. At the top of the
large loop are three bases, the anticodon, which is the complement of the
codon. There are 61 different tRNAs, each having a different binding site for
the amino acid and a different anticodon. For the codon UUU, the
complementary anticodon is AAA. Amino acid linkage to the proper tRNA is
controlled by the aminoacyl-tRNA synthetases. Energy for binding the amino
acid to tRNA comes from ATP conversion to adenosine monophosphate
(AMP).

Two models of tRNA. Image from Purves et al., Life: The Science of Biology, 4th
Edition, by Sinauer Associates (www.sinauer.com) and WH Freeman
(www.whfreeman.com), used with permission.

Translation is the process of converting the mRNA codon sequences into an


amino acid sequence. The initiator codon (AUG) codes for the amino acid Nformylmethionine (f-Met). No transcription occurs without the AUG codon. fMet is always the first amino acid in a polypeptide chain, although frequently
it is removed after translation. The intitator tRNA/mRNA/small ribosomal
unit is called the initiation complex. The larger subunit attaches to the
initiation complex. After the initiation phase the message gets longer during
the elongation phase.

Translation. Image from Purves et al., Life: The Science of Biology, 4th Edition, by
Sinauer Associates (www.sinauer.com) and WH Freeman (www.whfreeman.com), used
with permission.

New tRNAs bring their amino acids to the open binding site on the
ribosome/mRNA complex, forming a peptide bond between the amino acids.
The complex then shifts along the mRNA to the next triplet, opening the A
site. The new tRNA enters at the A site. When the codon in the A site is a
termination codon, a releasing factor binds to the site, stopping translation and
releasing the ribosomal complex and mRNA.

Termination. Image from Purves et al., Life: The Science of Biology, 4th Edition, by
Sinauer Associates (www.sinauer.com) and WH Freeman (www.whfreeman.com), used
with permission.

Often many ribosomes will read the same message, a structure known as a
polysome forms. In this way a cell may rapidly make many proteins.

Many ribosomes translating the same message, a polysome. Image from Purves
et al., Life: The Science of Biology, 4th Edition, by Sinauer Associates
(www.sinauer.com) and WH Freeman (www.whfreeman.com), used with permission.

The illustration below is from Genentech's Access Excellence site, which may
be reeached by clicking here. The drawing is available at
http://www.gene.com/ae/AB/GG/protein_synthesis.html

Mutations Redefined | Back to Top


We earlier defined mutations as any change in the DNA. We now can refine
that definition: a mutation is a change in the DNA base sequence that results
in a change of amino acid(s) in the polypeptide coded for by that gene. Alleles
are alternate sequences of DNA bases (genes), and thus at the molecular level
the products of alleles differ (often by only a single amino acid, which can
have a ripple effect on an organism by changing ). Addition, deletion, or
addition of nucleotides can alter the polypeptide. Point mutations are the result
of the substitution of a single base. Frame-shift mutations occur when the
reading frame of the gene is shifted by addition or deletion of one or more
bases. With the exception of mitochondria, all organisms use the same genetic
code. Powerful evidence for the common ancestry of all living things.

Links | Back to Top

Protein Synthesis Cartoon by Millard Susman (University of Wisconsin)


Download an animation of the process (sorry, you will need a Macintosh).
Animated GIF of Translation From the Gene Zine.
Amino Acid Anatomy Cartoons and animations on the basics of amino acids.
Amino Acids Linear formulae and links to images of the twenty amino acids
common to all life (at least as we know it).
Protein Synthesis Slideshow (West Georgia College) Slideshow and
downloadable animation of the process.
Ribosomes A text with links to illustrations. More than you ever wanted to know
about ribosomes!
Protein Synthesis A series of drawings (all on one web page) illustrating the
process.
The Genetic Code (Whitman College) and modifications.
The Genetic Code (U Texas Med. Center, Tyler)
Codon Usage Database Search for your favorite organism and check out its
genetic code.
DNA and Protein Synthesis (University of Illinois) Nice graphics and text as
well as links to additional resources. The color drawings above are from this
site.
The RNA World (IMB Jena, Germany) Links to WWW RNA sites and
resources. Lots of very cool images.

Text 1992, 1994, 1997, 1998, 2000, 2001, by M.J. Farabee, all rights reserved. Use for
educational purposes is encouraged.
Back to Table of Contents |

Email: mj.farabee@emcmail.maricopa.edu
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