What do ribozymes catalyze

Segments of these latter introns are transcribed within two or even three separate molecules from distant regions of the genome and the exons are assembled by a trans -splicing reaction reviewed by [53]. As yet, no tertiary model has been proposed. This suggests that there are two independent reaction centers or that there is a single site that switches substrates [16].

The reaction can also be initiated, both in vitro and in vivo, by the nucleophilic attack of water [ 48 , 54 ], although this is not a typical route. In this case, the intron is released as a linear molecule. As mentioned above, these reactions can also be catalyzed in trans from separate transcripts.

The splicing mechanism and the lariat product are reminiscent of those found in nuclear mRNA splicing, and it provides an additional evolutionary link between the two splicing reactions [ 23 , 47 ].

The applications of group II introns are more limited than those of group I. Nevertheless, it can catalyze the cleavage of ligated precursors using the energy of a phosphoanhydride bond, ligate RNA to DNA, and cleave single-stranded DNA substrates [55]. It is also used to circularize exon sequences [56] , as described above for group I introns. However, the fact that the intron RNA becomes inserted into double-stranded DNA during intron homing [ 24 , 25 ] suggests that other applications will soon be found.

The basic protein is essential for activity in vivo, however the RNase P RNA, by itself, can catalyze the reaction in vitro [2]. This reaction requires high salt concentrations e. However, the protein also affects cleavage-site specificity and turnover, so its full role in the reaction is still unclear reviewed by [ 58 , 60 ].

RNase P from E. RNase P RNA is the only characterized ribozyme that, unmodified, acts in trans on multiple substrates, and hence it is considered the only true, naturally occurring, RNA enzyme. RNase P from the nuclei and mitochondria of eukaryotes also exist as ribonucleoprotein complexes, although they generally have much higher protein contents ca. Archaebacteria have many properties that make them more similar to the eukaryotes than to the eubacteria, and they likewise are not known to have RNA-alone catalytic activity [60].

There is evidence that another ribonuclease in eukaryotes, RNase MRP, is closely related, and perhaps homologous i. It is a ribonucleoprotein complex that participates in nucleolar pre-rRNA processing. Although there is little sequence conservation, all the eubacterial RNase P RNAs can be folded into similar, although not identical, secondary structures on the basis of comparative sequence analyses [ 60 , 65 , 66 ].

The E. These two RNAs are the most extensively studied, but through a careful comparative analysis of the different eubacteria, it is possible to derive a common core structure consisting of helices P1—P5, P7—P12 and P15 [60]. The black spheres represent invariant nucleotides at the catalytic site and conserved nucleotides in the T-loop recognition site. Note that these conserved residues are clustered close to each other in what forms the catalytic core of the molecule. This figure is modified from [71].

The EGS, which is used to define the target specificity, is shown bound to the substrate boxed and the cleavage site is indicated by an arrow. While probably not needed for catalysis, these structures nevertheless lower the ionic strength requirements and enhance their thermal stability [60].

Such elements may be redundant in the sense that any single one can be deleted or modified without significantly altering the activity, but it is not possible to simultaneously delete or modify all of them. The RNase P RNA from eukaryotes and archaebacteria have little sequence similarity to their eubacterial counterparts, but they can often be folded into a universally conserved core structure as well [67].

Missing RNA elements from the structures may be supplanted by the protein component s , but this has not been characterized. Three-dimensional models of the E. Three-dimensional models for the B.

These are computer-derived models based on data obtained from phylogenetic comparisons, mutational analyses, chemical probing and from crosslinking studies.

It is beyond the scope of this review to discuss these models, especially in light of their speculative nature, but generally the models have a good fit to the experimental data. However, many of the specific interactions vary; thus these models are expected to undergo continual refinement as additional experimental data become available.

RNase P uses water as a nucleophile to cleave the phosphodiester bond Fig. The exact mechanism by which the tRNA precursor is bound is still unclear. The primary sequence does not seem to be important nor does any single element uniquely define the cleavage site. Thus, recognition is largely, if not entirely, based on tertiary interactions with the substrate. The target sequence can be virtually anything. This reaction works in vitro, in bacteria and in human cells [72].

This increases the efficiency of the reaction, and it is used to inactivate thymidine kinase mRNA from herpes simplex virus in cell lines [73]. Yet, while the therapeutic potential of RNase P has been demonstrated, it has not been widely used in therapeutic applications.

However, they are also found within satellite RNAs of salamanders, Neurospora , and within another pathogenic satellite virus found in man. The viroid and satellite RNAs are generally replicated by an RNA-dependent rolling-circle mechanism, and the catalytic domains are thought to process the linear concatemers that are generated into unit-length progeny.

The linear, unit-length progenies produced during replication in vivo are subsequently ligated to form closed-circular molecules that are used in the next round of rolling-circle replication. It is reasonable to expect that this is catalyzed by the ribozyme as well since, mechanistically, it represents the reverse of the cleavage reaction, and it would be analogous to the splicing reaction carried out by the group I and group II introns. However, in vitro only the hairpin ribozyme shows significant ligation activity.

Protein factors may be involved in vivo or other, as yet unidentified, RNA elements may be required. The catalytic domains of these ribozymes are small and relatively well characterized, and they are more widely used in therapeutic applications. Each has characteristics that confer specific advantages and disadvantages as therapeutic agents.

The in vitro and ex vivo activity of cis -cleaving forms of three of these self-cleaving ribozymes hammerhead, hairpin and HDV have been compared [75].

The hammerhead ribozyme is probably the most extensively studied of all the ribozymes, and it is the motif most commonly found in the viroids and satellite RNAs reviewed by [13]. Currently 16 hammerhead motifs are known in the plus and minus strands of these plant pathogens.

Three other hammerhead motifs are found in the satellite 2 RNAs from the salamanders, Triturus vulgaris, Ambystoma talpoideum and Amphiuma tridactylum [13] and references therein. This ribozyme was so named because its Australian discoverers found the secondary structure, as originally drawn, to be reminiscent of the head of a hammerhead shark. It is the smallest of the naturally occurring self-cleaving RNAs, at 40—50 nucleotides in length.

Analyses of the hammerhead ribozyme are voluminous reviewed by [ 9 , 76—78 ]. It consists of three helical regions, which are variable, and three single-stranded regions that contain most of the highly conserved nucleotides Fig. The length of the helical arms can be quite variable, and helix II can be reduced to two base pairs. Mutating any of the conserved residues markedly reduces activity; consequently, important functional groups are often identified by incorporating synthetic nucleotide analogs into the RNA e.

Characteristics of the hammerhead ribozyme. The dots represent nucleotides that can be anything, Y is a pyrimidine, R is a purine and H is any nucleotide except guanosine.

The arrow indicates the self-cleavage site. The boxed region shows the portion that is normally the substrate in trans -cleaving versions of the ribozyme. The numbering is based on standardized nomenclature []. This figure is modified from [81].

Cleavage occurs after an NUH triplet, where N is any nucleotide, and H is any nucleotide except guanosine. The most effective triplet is GUC, but other triplet combinations will work nearly as well; their relative activities have been compared, although the ordering can vary depending on the method of analysis reviewed by [ 8 , 9 , 77 ]. The reaction products are consistent with an S N 2 in-line reaction mechanism; this was suggested by an inversion of the phosphate at the scissile linkage.

The hammerhead ribozyme was the first catalytic RNA for which the complete X-ray crystal structure was solved. There are now a number of such structures available with resolutions ranging from 2.

The molecule has a Y shape Fig. This latter structure is more compatible with an S N 2 reaction mechanism. This is in contrast to the previously solved structures that showed the ribozyme in a ground state that was incompatible with such a mechanism. However, the existence of this site is still debated see [78]. Moreover, there are other incompatibilities between the experimental data and the crystalline structures [78] , and clearly additional work will be needed.

The hybridizing arms are varied to optimize ribozyme activity and substrate specificity. Normally hybridizing arms of six or seven base pairs are considered optimal, but for variable arms it is better to have a long stem III and short stem I than the reverse reviewed by [9]. The malleability of the hammerhead ribozyme makes it the most commonly used ribozyme for in vivo studies, and there are many successful examples reviewed by [ 7—9 ].

The hairpin ribozyme is found in three pathogenic, plant, satellite viruses, although the one found in the satellite virus associated with tobacco ring spot virus sTRSV is the best characterized. It consists of two noncontiguous sequences of 50 and 14 nucleotides within the minus strand of sTRSV reviewed by [ 13 , 17 , 82—84 ]. The secondary structure was determined based on computer-aided modeling, limited phylogenetic comparisons, mutational analyses and by in vitro selection.

Secondary structure of the hairpin ribozyme. The arrow shows the cleavage site. The boxed region represents the portion that is normally the substrate in trans -cleaving reactions. This figure is modified from [83] and it incorporates recent experimental data from [].

The other hairpin ribozymes are found in the satellite viruses of arabis mosaic virus sARMV and chicory yellow mottle virus sCYMV , and they mostly differ from the sequence shown in Fig. Indeed, the mutational and in vitro selection analyses show that the helical regions are structural elements that can largely be changed, as long as the integrity of the helices is maintained. Most of the conserved nucleotides occur within the single-stranded regions. However, the role this motif plays in catalysis is still unknown.

The consensus sequence and structure are shown in Fig. Recently, a computer-generated tertiary model was made that was based on preexisting structural data and on the spatial distance of tolerated, interdomain, aryl-disulfide crosslinks [85]. Additional information was also obtained by Walker et al. This places the two highly conserved bulged regions in proximity, and they could thus form the catalytic core. However, this tertiary model is still preliminary and additional data are required before the details of the catalytic site are known.

A major advantage of the hairpin ribozyme lies in its ability to catalyze both cleavage and ligation reactions efficiently in vitro; this has greatly facilitated in vitro selection experiments because new substrates, with the appropriate PCR primer sites, are easily generated [82] and references therein. The RNA-catalyzed ligation reaction is also thought to be relevant in vivo, in that the RNA can both cleave the linear multimers generated during rolling-circle replication and ligate them to form the circular RNA progeny.

However, as with the other catalytic motifs used in viroid replication, the cleavage-ligation reaction must be carefully regulated in vivo to prevent inappropriate cleavage, or ligation, of the resulting progeny. The mechanism by which this is accomplished is still unknown. Like the other catalytic RNAs, the hairpin ribozyme reaction requires a divalent cation. Stem II should be four base pairs, but stem I can be significantly extended e.

Substrate specificity is changed by altering the nonconserved residues within the base-paired region. The hairpin ribozyme is used to target HIV-1 RNA in cell culture, and it is currently approved for clinical trials see [84]. However, its effective use as a trans -cleaving ribozyme in vivo is still rather limited; the reasons for this are unclear. These monomers are then reverse transcribed and made double stranded to form the mature VS plasmid. In vitro transcribed VS RNA precursors are cleaved and ligated by the RNA itself and this is presumed to occur in vivo as well [90] and references therein.

At nucleotides long it is also the largest. However, this structure can be reduced to — nucleotides by making internal deletions within the helices [91]. An RNA secondary structure is proposed, but except for a tertiary interaction between loop I and loop V, little is known about its overall conformation Fig. The trans -cleaving form consists of nucleotides — and the substrate is shown boxed. As with RNase P, the ribozyme seems to recognize the structure of the substrate largely as a helical domain.

However, the uncertainty in the substrate requirements and the lackadaisical activity in trans have limited its application, although recent experiments have improved its activity [93]. The hepatitis delta virus HDV is a viroid-like satellite virus of the hepatitis B virus HBV , and it is the sole example of such a virus in mammalian systems reviewed by [ 94 , 95 ].

It is widespread and can cause severe fulminant hepatitis in infected patients. It is about nucleotides long, and it encodes a single protein that is expressed in two forms due to an RNA editing event.

Both the genomic, infectious strand, and the antigenomic strand have self-cleaving domains reviewed by [ 96—99 ]. Despite previous pronouncements, no biologically relevant, RNA-catalyzed, ligation reaction has been observed in vitro, although the integrity of the RNA catalytic domains is clearly essential for both the cleavage and ligation reaction in vivo [99] and references therein.

A possible mechanism for the biological control of these reactions, to prevent inappropriate cleavage or ligation, has been proposed [96]. Despite the sequence differences, both sequences fold into similar secondary structures, of which the pseudoknotted structure shown in Fig. The catalytic domains of HDV are known for their ability to retain cis -cleaving activity at high temperatures and in the presence of denaturants see [97] and references therein.

The tertiary structures have been computer modeled for the genomic and antigenomic forms of the pseudoknot model [ , ]; Fig. Characteristics of the HDV ribozyme. Numbering of the nucleotides is relative to the cleavage sites, indicated by arrows. Helical domains are separated by lines to facilitate the presentation. The identities of the nonbase-paired residues in the substrate boxed are not important for trans -cleaving activity.

Additional base-pair interactions, that were recently derived for the genomic ribozyme, are shown as dashed lines.

This figure is modified from []. Recently, the crystal structure for the genomic HDV ribozyme was obtained []. This structure is similar to that shown here except that there is an additional pseudoknot interaction between C21 and C22 with G38 and G Recently, a 2. This was accomplished by replacing hairpin IV with a small hairpin structure that binds tightly to the protein U1A, a spliceosomal protein. By co-crystallizing the RNA with the protein, the authors were better able to obtain highly structured crystals that diffracted to a high resolution.

It also greatly facilitated heavy metal substitution that is necessary for obtaining crystal phasing. This structure is very similar to the computer-predicted model, but it revealed some unexpected results. These specific interactions were not previously predicted, although the importance of the nucleotides were correctly derived [] In addition, A43 and G74 stack on the end of helix IV to form noncanonical base pairs and G10 forms an extension to helix II as previously predicted [ 97 , 98 ].

These interactions create a structure where helix IV is rotated relative to the computer model and hairpin III is more compressed. Nevertheless, the two structures are otherwise very similar. The crystal structure provides an organized, almost protein-like, crevice for the active site.

As with the other ribozymes, the cis -cleaving activity of the HDV ribozymes can be converted into a trans -cleaving activity reviewed by [ 97—99 ]. The most common form is indicated in Fig. Since most of the conserved elements are contained within the substrate, the practical utility of this latter form is somewhat limited. A completely closed-circular variant of the trans -cleaving ribozyme shown in Fig.

The absence of free ends makes this ribozyme particularly resistant to the exonucleases found in serum and the cellular environment. It is possible to change the substrate binding sequence to target other RNAs. However, in practice many of the changes in the substrate-binding sequence have unpredictable effects.

Up to now, the HDV ribozyme has a rather limited use in therapeutic applications, largely because of the difficulty of obtaining high activity with the trans -cleaving forms. This has applications, for example, in processing ribozyme cassettes transcripts containing multiple ribozyme units , for generating homogeneous ends on in vitro transcribed RNA or for the expression of discrete viral RNA transcripts off plasmid DNA [99] and references therein.

In this review I have attempted to summarize briefly the important features of the different catalytic RNAs that have so far been identified. The ribozyme field has advanced far in an incredibly short time. There are seven ribozymes identified in nature, and all of them have been engineered to cleave or modify other RNAs in trans.

Other ribozymes have been created de novo, and they can catalyze a variety of reactions. Moreover, other cellular processes have significant RNA components e. Additional ribozymes are bound to be discovered in the future.

Cited By Google Scholar. Ahead-of-Issue article Alert. Latest Issue Alert. Submit your work. Regulation and functional role of the electron transport chain supercomplexes. Structural insights into the interactions of Polycomb Repressive Complex 2 with chromatin.

TOP2B's contributions to transcription. GB Close Modal. This site uses cookies. By continuing to use our website, you are agreeing to our privacy policy. In , Carl Woese , Francis Crick , and Leslie Orgel were the first to suggest that RNA could act as a catalyst based upon findings that it can form complex secondary structures. In , Thomas R. 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. RNA can also act as a hereditary molecule, which encouraged Walter Gilbert to propose that in the 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. 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, artificially-produced self-cleaving RNAs that have good enzymatic activity have been produced. Katunin, V. Important contribution to catalysis of peptide bond formation by a single ionizing group within the ribosome.

Cell 10 , — Youngman, E. The active site of the ribosome is composed of two layers of conserved nucleotides with distinct roles in peptide bond formation and peptide release. Cell , — Pedersen, S. Escherichia coli ribosomes translate in vivo with variable rate. Weinger, J. Substrate-assisted catalysis of peptide bond formation by the ribosome. Winkler, W. Control of gene expression by a natural metabolite-responsive ribozyme.

Teixeira, A. Xu, R. Atomic structure of PDE4: insights into phosphodiesterase mechanism and specificity. Mildvan, A. Structures and mechanisms of Nudix hydrolases. Huai, Q. Schmeing, T. A pre-translocational intermediate in protein synthesis observed in crystals of enzymatically active 50S subunits.

Download references. The authors thank E. Friedman, K. Karbstein and A. Ke for helpful comments on the manuscript, R. Green and A. Mildvan for stimulating discussions, and A. Ke and S. Dorner for preparation of Figures 1b,d , 2b,d and 3b. You can also search for this author in PubMed Google Scholar. Reprints and Permissions. Doudna, J. Ribozyme catalysis: not different, just worse.

Nat Struct Mol Biol 12, — Download citation. Received : 06 January Accepted : 05 April Published : 03 May Issue Date : 01 May Anyone you share the following link with will be able to read this content:. Sorry, a shareable link is not currently available for this article. Provided by the Springer Nature SharedIt content-sharing initiative. Topics in Current Chemistry Scientific Reports Biophysical Reviews Journal of Molecular Evolution Nature Chemical Biology Advanced search.

Skip to main content Thank you for visiting nature. Abstract Evolution has resoundingly favored protein enzymes over RNA-based catalysts, yet ribozymes occupy important niches in modern cell biology that include the starring role in catalysis of protein synthesis on the ribosome.

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Figure 3: Possible mechanism for peptide bond formation catalyzed by the large subunit of the ribosome. References 1 Jencks, W. Google Scholar 2 Narlikar, G. Google Scholar 16 Murray, J. Acknowledgements The authors thank E. View author publications. Ethics declarations Competing interests The authors declare no competing financial interests. Rights and permissions Reprints and Permissions. About this article Cite this article Doudna, J. Copy to clipboard. Anthony M.

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