Ribozymes

Until about 20 years ago, all known enzymes were proteins. But then it was discovered that some RNA molecules can act as enzymes; that is, catalyze covalent changes in the structure of substrates (most of which are also RNA molecules). Catalytic RNA molecules are called ribozymes. Most classes of RNA

transfer RNA (tRNA) ribosomal RNA (rRNA) messenger RNA (mRNA)

are transcribed as precursors that are larger than the final product. These precursors often contain

"head" (5') and "tail" (3') sequences intron sequences

that must be removed to make the final product. Some of the processing steps employ other RNA molecules (always associated with proteins).

Ribonuclease P
All living things synthesize an enzyme called Ribonuclease P (RNase P) that cleaves the head (5') end of the precursors of transfer RNA (tRNA) molecules. In bacteria, ribonuclease P is a heterodimer containing

a molecule of RNA and one of protein

Separated from each other, the RNA retains its ability to catalyze the cleavage step (although less efficiently than the intact dimer), but the protein alone cannot do the job.

Group I Introns
Some ribosomal RNA (rRNA) genes

in the mitochondrial genome of certain fungi (e.g., yeast) in some chloroplast genomes in the nuclear genome of some "lower" eukaryotes, for example o the ciliated protozoan Tetrahymena thermophila) o the plasmodial slime mold Physarum polycephalum

contain introns that must be spliced out to make the final product.

The splicing reaction is self-contained; that is, the intron - with the help of associated proteins - splices itself out of the precursor RNA. Once excision of the intron and splicing of the adjacent exons are completed, the story is over. In other words, although the action is catalyzed by the RNA, only a single molecule of substrate is involved (unlike protein enzymes that repeatedly catalyze a reaction). However, synthetic versions of Group I introns made in the laboratory can - in vitro - act repeatedly; that is, like true enzymes. The DNA of some Group I introns includes an open reading frame (ORF) that encodes a transposase-like protein that can make a copy of the intron and insert it elsewhere in the genome. All the Group I introns share a characteristic secondary structure and mode of action that distinguishes them from the next group.

Group II Introns
Some messenger RNA (mRNA) genes

in the mitochondrial genome of yeast and other fungi (encoding the proteins cytochrome b and subunits of cytochrome c oxidase) in some chloroplast genomes

also contain self-splicing introns. Because their secondary structure and the details of the splicing reaction differ from the rRNA introns discussed above, these are called Group II introns. The DNA of some Group II introns also includes an open reading frame (ORF) that encodes a transposase-like protein that can make a copy of the intron and insert it elsewhere in the genome.

Spliceosomes
Spliceosomes remove introns and splice the exons of most nuclear genes. They are composed of 5 kinds of small nuclear RNA (snRNA) molecules and a large number of protein molecules. It is the snRNA not the protein that catalyzes the splicing reactions. The molecular details of the reactions are similar to those of Group II introns, and this has led to speculation that this splicing machinery evolved from them.

Viroids
Viroids are

RNA molecules that infect plant cells as conventional viruses do, but o are far smaller (one has only 246 nucleotides) o are naked; that is, they are not encased in a capsid.

Some viroidlike molecules get into the cell as passengers inside a conventional plant virus. These are called virusoids or viroidlike satellite RNAs. In both cases, the molecules consists of o single-stranded RNA whose o ends are covalent bonded to form a circle. o There are several regions where base-pairing occurs across adjacent portions of the molecule. New viroids and virusoids are synthesized by the host cell as long precursors in which the viroid structure is tandemly repeated. These repeats must be cut out and ligated to form the final product. Most virusoids and at least one viroid are self-splicing; that is they can cut themselves out of the precursor and ligate their ends without the aid of any host enzymes. Thus they represent another class of ribozyme.

Both viroids and virusoids are responsible for a number of serious diseases of economically important plants; e.g. the coconut palm and chrysanthemums. (The problem is so severe with chrysanthemums that all growers in the U.S. now secure their stock from a few companies that raise the plants in "clean" rooms using stringent precautions to prevent infection by the viroid.)

The Ribosome is a Ribozyme

Ribosomes are huge aggregates containing 3 (4 in eukaryotes) rRNA molecules and scores of protein molecules. The three-dimensional structure of the large (50S) subunit of a bacterial ribosome was published in August 2000. It clearly shows that formation of the peptide bond that links each amino acid to the growing polypeptide chain is catalyzed by the 23S RNA molecule in the large subunit. The 31 proteins in the subunit probably provide the scaffolding needed to maintain the tertiary structure of the RNA.

Ribozymes for Human Therapy

The ability of ribozymes to recognize and cut specific RNA molecules makes them exciting candidates for human therapy. Already, a synthetic ribozyme that destroys the mRNA encoding a receptor of Vascular Endothelial Growth Factor (VEGF) is being readied for clinical trials. VEGF is a major stimulant of angiogenesis, and blocking its action may help starve cancers of their blood supply.

Ribozyme
Structure of hammerhead ribozyme A ribozyme (from ribonucleic acid enzyme, also called RNA enzyme or catalytic RNA) is an RNA molecule possessing a well defined tertiary structure that enables it to catalyze a chemical reaction. Many natural ribozymes catalyze either the hydrolysis of one of their own phosphodiester bonds, or the hydrolysis of bonds in other RNAs, but they have also been found to catalyze the aminotransferase activity of the ribosome. Investigators studying the origin of life have produced ribozymes in the laboratory that are capable of catalyzing their own synthesis under very specific conditions, such as an RNA polymerase ribozyme. Mutagenesis and selection has been performed resulting in isolation of improved variants of the "Round-18" polymerase ribozyme from 2001. "B6.61" is able to add up to 20 nucleotides to a primer template in 24 hours, until it decomposes by hydrolysis of its phosphodiester bonds. Some ribozymes may play an important role as therapeutic agents, as enzymes which tailor defined RNA sequences, as biosensors, and for applications in functional genomics and gene discovery.

Schematic showing ribozyme cleavage of RNA. Before the discovery of ribozymes, enzymes, which are defined as catalytic proteins, were the only known biological catalysts. In 1967, Carl Woese, Francis Crick, and Leslie Orgel were the first to suggest that RNA could act as a catalyst. This idea was based upon the discovery that RNA can form complex secondary structures. The first ribozymes were discovered in the 1980s by Thomas R. Cech, who was studying RNA splicing in the ciliated protozoan Tetrahymena thermophila and Sidney Altman, who was working on the bacterial RNase P complex. These ribozymes were found in the intron of an RNA transcript, which removed itself from the transcript, as well as in the RNA component of the RNase P complex, which is involved in the maturation of pre-tRNAs. In 1989, Thomas R. Cech and Sidney Altman won the Nobel Prize in chemistry for their "discovery of catalytic properties of RNA." The term ribozyme was first introduced by Kelly Kruger et al. in 1982 in a paper published in Cell. It had been a firmly established belief in biology that catalysis was reserved for proteins. In retrospect, catalytic RNA makes a lot of sense. This is based on the old question regarding the origin of life: Which comes first, enzymes that do the work of the cell or nucleic acids that carry the information required to produce the enzymes? Nucleic acids as catalysts circumvents this problem. In the 1970s Thomas Cech, at the University of Colorado at Boulder, was studying the excision of introns in a ribosomal RNA gene in Tetrahymena thermophila. While trying to purify the enzyme responsible for splicing reaction, he found that intron could be 4

spliced out in the absence of any added cell extract. As much as they tried, Cech and his colleagues could not identify any protein associated with the splicing reaction. After much work, Cech proposed that the intron sequence portion of the RNA could break and reform phosphodiester bonds. At about the same time, Sidney Altman, who is a Professor at Yale University, was studying the way tRNA molecules are processed in the cell when he and his colleagues isolated an enzyme called RNase-P, which is responsible for conversion of a precursor tRNA into the active tRNA. Much to their surprise, they found that RNase-P contained RNA in addition to protein and that RNA was an essential component of the active enzyme. This was such a foreign idea that they had difficulty publishing their findings. The following year, Altman demonstrated that RNA can act as a catalyst by showing that the RNase-P RNA subunit could catalyze the cleavage of precursor tRNA into active tRNA in the absence of any protein component. Since Cech's and Altman's discovery, other investigators have discovered other examples of self-cleaving RNA or catalytic RNA molecules. Many ribozymes have either a hairpin or hammerhead shaped active center and a unique secondary structure that allows them to cleave other RNA molecules at specific sequences. It is now possible to make ribozymes that will specifically cleave any RNA molecule. These RNA catalysts may have pharmaceutical applications. For example, a ribozyme has been designed to cleave the RNA of HIV. If such a ribozyme was made by a cell, all incoming virus particles would have their RNA genome cleaved by the ribozyme, which would prevent infection.

Activity
Although most ribozymes are quite rare in the cell, their roles are sometimes essential to life. For example, the functional part of the ribosome, the molecular machine that translates RNA into proteins, is fundamentally a ribozyme, composed of RNA tertiary structural motifs that are often coordinated to metal ions such as Mg2+ as cofactors. There is no requirement for divalent cations in a five-nucleotide RNA that can catalyze transphenylalanation of a four-nucleotide substrate which has three base complementary sequence with the catalyst. The catalyst and substrate were devised by truncation of the C3 ribozyme. RNA can also act as a hereditary molecule, which encouraged Walter Gilbert to propose that in the distant past, the cell used RNA as both the genetic material and the structural and catalytic molecule, rather than dividing these functions between DNA and protein as they are today. This hypothesis became known as the "RNA world hypothesis" of the origin of life. If ribozymes were the first molecular machines used by early life, then today's remaining ribozymes -- such as the ribosome machinery -- could be considered living fossils of a life based primarily on nucleic acids. A recent test-tube study of prion folding suggests that an RNA may catalyze the pathological protein conformation in the manner of a chaperone enzyme.

Artificial ribozymes
Since the discovery of ribozymes that exist in living organisms, there has been interest in the study of new synthetic ribozymes made in the laboratory. For example, artificiallyproduced self-cleaving RNAs that have good enzymatic activity have been produced. Some of the synthetic ribozymes that were produced had novel structures, while some were similar to the naturally occurring hammerhead ribozyme. The techniques used to discover artificial ribozymes involve Darwinian evolution. This approach takes advantage of RNA's dual nature as both a catalyst and an informational polymer, making it easy for an investigator to produce vast populations of RNA catalysts using polymerase enzymes. The ribozymes are mutated by reverse transcribing them with reverse transcriptase into various cDNA and amplified with mutagenic PCR. The selection parameters in these experiments often differ. One approach for selecting a ligase ribozyme involves using biotin tags, which are covalently linked to the substrate. If a molecule possesses the desired ligase activity, a streptavidin matrix can be used to recover the active molecules. Lincoln and Joyce developed an RNA enzyme system capable of self replication in about an hour. By utilizing molecular competition (in vitro evolution) of a candidate enzyme mixture, a pair of RNA enzymes emerged, in which each synthesizes the other from synthetic oligonucleotides, with no protein prese

Hairpin ribozyme

Secondary structure of a minimal hairpin ribozyme with substrate RNA bound. The hairpin ribozyme is a small section of RNA that can act as an enzyme known as a ribozyme. Like the hammerhead ribozyme it is found in RNA satellites of plant viruses. It was first identified in the minus strand of the tobacco ringspot virus (TRSV) satellite RNA where it catalyzes self-cleavage and joining (ligation) reactions to process the products of rolling circle virus replication into linear and circular satellite RNA molecules. The hairpin ribozyme is similar to the hammerhead ribozyme in that it does not require a metal ion for the reaction. The hairpin ribozyme has been identified in only 3 naturally occurring sequences:

satellite RNA of tobacco ringspot virus (sTRSV) satellite RNA of chicory yellow mottle virus (sCYMV) satellite RNA of arabis mosaic virus (sARMV)

Smaller artificial versions of the hairpin ribozyme have been developed to enable a more detailed experimental analysis of the molecule.This is a commonly used strategy for separating those parts of a self-processing RNA molecule that are essential for the RNA processing reactions from those parts which serve unrelated functions. This strategy was important in that it allowed investigators to (i) apply biochemical methods for enzymatic analysis, (ii) conduct experiments to identify essential structural elements of the ribozyme-substrate complex, and (iii) develop engineered ribozymes that have been used for biomedical applications, including preventing the replication of pathogenic viruses, and the study of the function of individual genes.

Biological function

The hairpin ribozyme is an RNA motif that catalyzes RNA processing reactions essential for replication of the satellite RNA molecules in which it is embedded. These reactions are self-processing, i.e. a molecule rearranging its own structure. Both cleavage and end joining reactions are mediated by the ribozyme motif, leading to a mixture of interconvertible linear and circular satellite RNA molecules. These reactions are important for processing the large multimeric RNA molecules that are generated by rolling circle replication. At the end of the replication cycle, these large intermediates of satellite RNA replication are processed down to unit length molecules (circular or linear) before they can be packaged by viruses and carried to other cells for further rounds of replication.

Folding of the hairpin ribozyme in its native tertiary structure. The ribozyme sequence is shown in grey, whilst the substrate sequence is light red. The cleavage and ligation site (dark red) is between nucleotides A-1 and G+1. Important sequences within loops A and 8

B are shown, with black dots indicating non-Watson-Crick interactions between nucleotides. The two catalytic nucleotides are shown in green, and the critical nucleotide C25, which forms a Watson-Crick base pair with G+1 at the reaction site, is shown in blue.

Reaction chemistry
In common with several other ribozymes and protein ribonucleases, the cleavage reaction of the hairpin ribozyme generates RNA fragments with termini consisting of a 3',5'-cyclic phosphate and a 5'-hydroxyl group. The ligation reaction appears to be a simple reveral of cleavage, i.e. covalent joining of RNA fragments ending with a 3',5'-cyclic phosphate and a 5'-hydroxyl group to generate the ordinary 3'-5' phosphodiester linkage used in both RNA and DNA. Studies of this reaction in multiple ribozymes have served to establish that the reaction chemistry (catalytic mechanism) is an endogenous property of the RNA molecule itself and is not mediated by metal ions, as is true for some protein enzymes and some other ribozymes.

Targeted RNA cleavage and antiviral activity

Hairpin ribozymes have been modified in such a way that they can be used to target cleavage of other RNA molecules. This is possible because much of the substrate specificity of the hairpin ribozyme results from simple Watson-Crick base pairing within helices 1 and 2. One area of particular interest has been the development of hairpin ribozymes for potential therapeutic use, for example by preventing the replication of pathogenic viruses. Antiviral hairpin ribozymes have been generated and expressed within mammalian cells, and cells expressing different engineered ribozymes have been shown to be resistant to infection by HIV-1, hepatitis B, and Sindbis virus.

Hammerhead ribozyme
Hammerhead RNAs are RNAs that self-cleave via a small conserved secondary structural motif termed a hammerhead because of its shape. Most hammerhead RNAs are subsets of two classes of plant pathogenic RNAs: the satellite RNAs of RNA viruses and the viroids. The hammerhead sequence is sufficient for self-cleavage and acts by forming a conserved three-dimensional tertiary structure. In the natural state, a hammerhead RNA motif is a single strand of RNA, and although the cleavage is autocatalytic and takes place in the absence of protein enzymes, the hammerhead RNA itself is not a true enzyme in its natural state, as it cannot catalyze multiple turnovers. In vitro hammerhead constructs can be engineered such that they consist of two RNA strands. Such constructs are typically employed for in vitro experiments, and the term

"hammerhead RNA" has become in practice synonymous with the more frequently used "hammerhead ribozyme". The minimal hammerhead ribozyme sequence that is catalytically active consists of three base-paired stems flanking a central core of 15 conserved nucleotides. The conserved central bases, with few exceptions, are essential for ribozymes catalytic activity. The hammerhead ribozyme is arguably the best-characterized ribozyme. Its small size, thoroughly-investigated cleavage chemistry, known crystal structure, and its biological relevance make the hammerhead ribozyme particularly well-suited for biochemical and biophysical investigations into the fundamental nature of RNA catalysis. Hammerhead ribozymes may play an important role as therapeutic agents; as enzymes which tailor defined RNA sequences, as biosensors, and for applications in functional genomics and gene discovery. Hammerhead ribozymes are found in a wide range of plant viroids and helmenths such as:

Peach latent mosaic viroid Avocado sunblotch viroid Velvet tobacco mottle virus Satellite RNA Schistosoma mansoni Satellite DNA Dianthus caryophyllus viroid-like DNA Cherry small circular viroid-like RNA Newt Dolichopoda cave cricket

Biochemistry of catalysis
The hammerhead ribozyme carries out a very simple chemical reaction that results in the breakage of the substrate strand of RNA, specifically at C17, the cleavage-site nucleotide. Although RNA cleavage is often referred to as hydrolysis, the mechanism employed does not in fact involve the addition of water. Rather, the cleavage reaction is simply an isomerization that consists of rearrangement of the linking phosphodiester bond. It is the same reaction, chemically, that occurs with random base-mediated RNA degradation, except that it is highly site-specific and the rate is accelerated 10,000-fold or more.

Cleavage by phosphodiester isomerization, not hydrolysis

In-line transition-state for the hammerhead ribozyme reaction. A general base (B, red) abstracts a proton from the 2'-O, and a general acid (A, blue), supplies a proton to the 5'O leaving group as negative charge accumulates. The bonds breaking and forming (dotted lines) must be in the axial positions and reside approximately 180 apart, as shown. The reaction product is a 2',3'-cyclic phosphate. The cleavage reaction is a phosphodiester isomerization reaction that is initiated by abstraction of the cleavage-site ribose 2-hydroxyl proton from the 2-oxygen, which then becomes the attacking nucleophile in an in-line or SN2(P)-like reaction, although it is not known whether this proton is removed prior to or during the chemical step of the hammerhead cleavage reaction. (The cleavage reaction is technically not bimolecular, but 10

behaves in the same way a genuine SN2(P) reaction does; it undergoes inversion of configuration subsequent to forming an associative transition-state consisting of a pentacoordinated oxyphosphrane.) The attacking and leaving group oxygens will both occupy the two axial positions in the trigonal bipyramidal transition-state structure as is required for an SN2-like reaction mechanism. The 5-product, as a result of this cleavage reaction mechanism, possesses a 2,3-cyclic phosphate terminus, and the 3-product possesses a 5-OH terminus, as with nonezymatic alkaline cleavage of RNA. The reaction is therefore, in principle, reversible, as the scissile phosphate remains a phosphodiester, and may thus act as a substrate for hammerhead RNA-mediated ligation without a requirement for ATP or a similar exogenous energy source. The hammerhead ribozyme-catalyzed reaction, unlike the formally identical non-enzymatic alkaline cleavage of RNA, is a highly sequence-specific cleavage reaction with a typical turnover rate of approximately 1 molecule of substrate per molecule of enzyme per minute at pH 7.5 in 10 mM Mg 2+ (so-called standard reaction conditions for the minimal hammerhead RNA sequence), depending upon the sequence of the particular hammerhead ribozyme construct measured. This represents an approximately 10,000-fold rate enhancement over the nonezymatic cleavage of RNA.

Requirement for divalent metal ions

All ribozymes were originally thought to be metallo-enzymes, in the sense that they were assumed to require the presence of divalent cations, such as Mg2+, for both folding and catalysis. It was presumed that hexahydrated magnesium ions, which exist in equilibrium with magnesium hydroxide, could play the roles of general acid and general base, in a way analogous to those played by two histidines in RNase A. An additional role for divalent metal ions has also been proposed in the form of electrostatic stabilization of the transition-state.

Not a metallo-enzyme
In 1998 it was discovered that the hammerhead ribozyme, as well as the VS ribozyme and hairpin ribozyme, do not require the presence of metal ions for catalysis, provided a sufficiently high concentration of monovalent cation is present to permit the RNA to fold. This discovery suggested that the RNA itself, rather than serving as an inert, passive scaffold for the binding of chemically active divalent metal ions, is instead itself intimately involved in the chemistry of catalysis. The latest structural results, described below, indeed confirm that two invariant nucleotides, G12 and G8, are positioned consistent with roles as the general base and general acid in the hammerhead cleavage reaction. Strictly speaking, therefore, the hammerhead ribozyme cannot be a metallo-enzyme.

Minimal hammerhead ribozyme

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The minimal hammerhead sequence that is required for the self-cleavage reaction includes approximately 13 conserved or invariant "core" nucleotides, most of which are not involved in forming canonical Watson-Crick base-pairs. The core region is flanked by Stems I, II and III, which are in general made of canonical Watson-Crick base-pairs but are otherwise not constrained with respect to sequence. The catalytic turnover rate of minimal hammerhead ribozymes is ~ 1/min (a range of 0.1/min to 10/min is commonly observed, depending upon the nonconserved sequences and the lengths of the three helical stems). Much of the experimental work carried out on hammerhead ribozymes has used a minimal construct. Type I & type III hammerhead RNA
Hammerhead ribozyme (type I)

Type: 2 structure: Seed alignment: Avg length: Avg identity:

Gene; ribozyme; Published; PubMed Bateman A 45.70 nucleotides 76.00%

Hammerhead ribozyme (type III)

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Type: Gene; ribozyme; 2 structure: Published[8] Seed alignment: Bateman A Avg length: 54.2 nucleotides Avg identity: 77% Structurally the hammerhead ribozyme is composed of three base paired helices, separated by short linkers of conserved sequences. These helices are called I, II and III. Hammerhead ribozymes can be classified into three types based on which helix the 5' and 3' ends are found in. If the 5' and 3' ends of the sequence contribute to stem I then it is a type I hammerhead ribozyme, and if the and 3' ends of the sequence contribute to stem III then it is a type III hammerhead ribozyme. Of the three possible topological types both type I and type III are common. It is not known if examples of the type II topology are found in nature.

Full-length hammerhead ribozyme

The full-length hammerhead ribozyme consists of additional sequence elements in stems I and II that permit additional tertiary contacts to form. The tertiary interactions stabilize the active conformation of the ribozyme, resulting in cleavage rates up to 1000-fold greater than those for corresponding minimal hammerhead sequences.

Therapeutic applications
Modified hammerhead ribozymes are being tested as therapeutic agents. Synthetic RNAs containing sequences complementary to the mutant SOD1 mRNA and sequences necessary to form the hammerhead catalytic structure are being studied as a possible

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therapy for amyotrophic lateral sclerosis. Work is also underway to find out whether they could be used to engineer HIV resistant lines of T-cells. The therapeutic use of trans-cleaving hammerhead ribozymes has been severely hampered by its low-level activity in vivo. The true catalytic potential of trans-cleaving hammerhead ribozymes may be recouped in vivo and therapeutic derivatives are likely to complement other nucleic acid hybridizing therapeutic strategies. Already there are hammerhead ribozymes which are close to clinical application.

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