Mechanisms of RNA catalysis

Ribozymes are RNA molecules that act as chemical catalysts. In contemporary cells, most known ribozymes carry out phosphoryl transfer reactions. The nucleolytic ribozymes comprise a class of five structurally-distinct species that bring about site-specific cleavage by nucleophilic attack of the 2'-O on the adjacent 3'-P to form a cyclic 2',3'-phosphate. In general, they will also catalyse the reverse reaction. As a class, all these ribozymes appear to use general acid-base catalysis to accelerate these reactions by about a million-fold. In the Varkud satellite ribozyme, we have shown that the cleavage reaction is catalysed by guanine and adenine nucleobases acting as general base and acid, respectively. The hairpin ribozyme most probably uses a closely similar mechanism. Guanine nucleobases appear to be a common choice of general base, but the general acid is more variable. By contrast, the larger ribozymes such as the self-splicing introns and RNase P act as metalloenzymes.     

Restriction in the cleavage activity of hammerhead ribozymes ensures ongoing evolution in prebiotic RNA world

Self-cleaving infectious RNAs found in many plant viruses and viroids can also cleave intrans and form hammerhead type secondary structure. It has been observed that the cleavage site must contain the triplet GUC. Also, in other cases, the sequence XUY holds good where X = A, C, G, U and Y = A, C, U but not G. The high electronegative nature of guanosine holds the key to its resistance to cleavage which does not allow hybrid formation between the ribozyme and substrate strands. Guanosine resistance to cleavage might have been the starting thrust for the evolution of a translational initiation codon from XUG. A hypothesis is proposed in this regard and its evolutionary consequences are discussed briefly. Presented at the National Symposium on Evolution of Life.     

Metal ion specificities for folding and cleavage activity in the Schistosoma hammerhead ribozyme

The effects of various metal ions on cleavage activity and global folding have been studied in the extended Schistosoma hammerhead ribozyme. Fluorescence resonance energy transfer was used to probe global folding as a function of various monovalent and divalent metal ions in this ribozyme. The divalent metals ions Ca²⁺, Mg²⁺, Mn²⁺, and Sr²⁺ have a relatively small variation (less than sixfold) in their ability to globally fold the hammerhead ribozyme, which contrasts with the very large difference (>10,000-fold) in apparent rate constants for cleavage for these divalent metal ions in single-turnover kinetic experiments. There is still a very large range (>4600-fold) in the apparent rate constants for cleavage for these divalent metal ions measured in high salt (2 M NaCl) conditions where the ribozyme is globally folded. These results demonstrate that the identity of the divalent metal ion has little effect on global folding of the Schistosoma hammerhead ribozyme, whereas it has a very large effect on the cleavage kinetics. Mechanisms by which the identity of the divalent metal ion can have such a large effect on cleavage activity in the Schistosoma hammerhead ribozyme are discussed.     

Inhibition of gene expression with ribozymes

Summary 1. Ribozymes can be designed to cleavein trans, i.e. several substrate molecules can be turned over by one molecule of the catalytic RNA. Only small molecular weight ribozymes, or small ribozymes, are discussed in this review with particular emphasis on the hammerhead ribozyme as this has been most widely used for the inhibition of gene expression by cleavage of mRNAs.2. Cellular delivery of the ribozyme is of crucial importance for the success of inhibition of gene expression by this methodology. Two modes of delivery can be envisaged, endogenous and exogenous delivery. Of the former several variants exist, depending on the vector used. The latter is still in its infancy, even though chemical modification has rendered such ribozymes resistant against degradation by serum nucleases without impairment of catalytic efficiency.3. Various successful applications of ribozymes for the inhibition of gene expression are discussed, with particular emphasis on HIV1 and cancer targets. These examples demonstrate the promise of this methodology.     

RNase P RNA mediated cleavage: substrate recognition and catalysis

The universally conserved endoribonuclease P consists of one RNA subunit and, depending on its origin, a variable number of protein subunits. RNase P is involved in the processing of a large variety of substrates in the cell, the preferred substrate being tRNA precursors. Cleavage activity does not require the presence of the protein subunit(s) in vitro. This is true for both prokaryotic and eukaryotic RNase P RNA suggesting that the RNA based catalytic activity has been preserved during evolution. Progress has been made in our understanding of the contribution of residues and chemical groups both in the substrate as well as in RNase P RNA to substrate binding and catalysis. Moreover, we have access to two crystal structures of bacterial RNase P RNA but we still lack the structure of RNase P RNA in complex with its substrate and/or the protein subunit. Nevertheless, these recent advancements put us in a new position to study the way and nature of interactions between in particular RNase P RNA and its substrate. In this review I will discuss various aspects of the RNA component of RNase P with an emphasis on our current understanding of the interaction between RNase P RNA and its substrate.     

The RNA World and Its Evolution

This paper develops Belozersky’s early idea of the precedence of RNA in the origin of life on the Earth. Based on the current knowledge of the functional omnipotence of RNA, three new mechanisms are considered that could be critical for the origin and evolution of the ancient RNA world: (1) the reaction of spontaneous transesterification of polyribonucleotides in aqueous media, which has been recently discovered by A.B. Chetverin and colleagues and could result in elongation of short initial oligoribonucleotides and generate sequence variants for further selection; (2) compartmentation of functional RNA ensembles in the form of mixed molecular colonies on moist mineral surfaces, in the absence of membranes and other envelopes; and (3) systematic exponential enrichment of an RNA population with “ functionally the best” molecules due to alternating dissolution of the colonies upon flooding and formation of new colonies upon drying in ancient pools (“primordial natural SELEX”).__________Translated from Molekulyarnaya Biologiya, Vol. 39, No. 4, 2005, pp. 550–556.Original Russian Text Copyright © 2005 by Spirin.     

A versatile communication module for controlling RNA folding and catalysis

To exert control over RNA folding and catalysis, both molecular engineering strategies and in vitro selection techniques have been applied toward the development of allosteric ribozymes whose activities are regulated by the binding of specific effector molecules or ligands. We now describe the isolation and characterization of a new and considerably versatile RNA element that functions as a communication module to render disparate RNA folding domains interdependent. In contrast to some existing communication modules, the novel 9-nt RNA element is demonstrated to function similarly between a variety of catalysts that include the hepatitis delta virus, hammerhead, X motif and Tetrahymena group I ribozymes, and various ligand-binding domains. The data support a mechanistic model of RNA folding in which the element is comprised of both canonical and non-canonical base pairs and an unpaired nucleotide in the active, effector-bound conformation. Aside from enabling effector-controlled RNA function through rational design, the element can be utilized to identify sites in large RNAs that are susceptible to effector regulation.     

RNA folding and catalysis mediated by iron (II)

Mg2? shares a distinctive relationship with RNA, playing important and specific roles in the folding and function of essentially all large RNAs. Here we use theory and experiment to evaluate Fe2? in the absence of free oxygen as a replacement for Mg2? in RNA folding and catalysis. We describe both quantum mechanical calculations and experiments that suggest that the roles of Mg2? in RNA folding and function can indeed be served by Fe2?. The results of quantum mechanical calculations show that the geometry of coordination of Fe2? by RNA phosphates is similar to that of Mg2?. Chemical footprinting experiments suggest that the conformation of the Tetrahymena thermophila Group I intron P4-P6 domain RNA is conserved between complexes with Fe2? or Mg2?. The catalytic activities of both the L1 ribozyme ligase, obtained previously by in vitro selection in the presence of Mg2?, and the hammerhead ribozyme are enhanced in the presence of Fe2? compared to Mg2?. All chemical footprinting and ribozyme assays in the presence of Fe2? were performed under anaerobic conditions. The primary motivation of this work is to understand RNA in plausible early earth conditions. Life originated during the early Archean Eon, characterized by a non-oxidative atmosphere and abundant soluble Fe2?. The combined biochemical and paleogeological data are consistent with a role for Fe2? in an RNA World. RNA and Fe2? could, in principle, support an array of RNA structures and catalytic functions more diverse than RNA with Mg2? alone.     

Folding of the hammerhead ribozyme: pyrrolo-cytosine fluorescence separates core folding from global folding and reveals a pH-dependent conformational change

The catalytic activity of the hammerhead ribozyme is limited by its ability to fold into the native tertiary structure. Analysis of folding has been hampered by a lack of assays that can independently monitor the environment of nucleobases throughout the ribozyme-substrate complex in real time. Here, we report the development and application of a new folding assay in which we use pyrrolo-cytosine (pyC) fluorescence to (1) probe active-site formation, (2) examine the ability of peripheral ribozyme domains to support native folding, (3) identify a pH-dependent conformational change within the ribozyme, and (4) explore its influence on the equilibrium between the folded and unfolded core of the hammerhead ribozyme. We conclude that the natural ribozyme folds in two distinct noncooperative steps and the pH-dependent correlation between core folding and activity is linked to formation of the G8-C3 base pair.     

Antiviral ribozymes

Catalytic RNAs are a genetic property not only of some particular viroids or viruses, but also are more common naturally among eukaryotes and even prokaryotes than earlier expected. However, the major interest in ribozymes results from their potential for development of “tailor-made” cDNA constructions designed to be transcribed into catalytic RNAs that will recognize by hybridization and destroy by specific cleavage their cellular or viral RNA targets. The efficiency of an antiviral ribozyme is determined by both the accessibility and sequence conservation of the target region, as well as the design of the ribozyme: its type, size, and composition of flanking sequences; expression rates; and cellular compartment localization. Until now the most frequently selected viral target is the human immunodeficiency virus, where an up to a 10⁴-fold inhibition in its progeny production has been achieved. Although the first generation ribozymes focused on improvements in basic design and expression rates, more recently the efficiency of antiviral catalytic activity has been increased by employing polyribozymes and/or multitarget ribozymes, as well as special constructions to enhance the cellular co-compartmentation of the ribozyme with its viral RNA target.     

RNA catalysis

Our understanding of the relationship between the structure of RNA and its catalytic activity has advanced significantly in the past year. These advances include time-resolved crystallographic studies on the hammerhead ribozyme, as well as new structures of a group I intron, a lead(II)-cleavage ribozyme, a hepatitis delta virus ribozyme, and components of the spliceosome machinery and the peptidyl transferase center of the ribosome and, most significantly, the structure of the ribosome itself.     

功能RNA分子的构建、表达及其在基因功能鉴定中的应用

人工构建的siRNAs、aptazymes、maxizymes以及intramers等功能RNA分子,可以在mRNA或蛋白质水平上调控基因的功能.功能RNA分子可在活体内或转基因模式动、植物中抑制目标基因的表达,使目标基因和蛋白质功能丧失,进而引起表型变异.胞内表达的活性RNAs可作为有效的研究工具应用于基因及其编码蛋白的功能鉴定,并在药物开发和人类疾病治疗上有潜在的应用前景.     

Role of tautomerism in RNA biochemistry

Heterocyclic nucleic acid bases and their analogs can adopt multiple tautomeric forms due to the presence of multiple solvent-exchangeable protons. In DNA, spontaneous formation of minor tautomers has been speculated to contribute to mutagenic mispairings during DNA replication, whereas in RNA, minor tautomeric forms have been proposed to enhance the structural and functional diversity of RNA enzymes and aptamers. This review summarizes the role of tautomerism in RNA biochemistry, specifically focusing on the role of tautomerism in catalysis of small self-cleaving ribozymes and recognition of ligand analogs by riboswitches. Considering that the presence of multiple tautomers of nucleic acid bases is a rare occurrence, and that tautomers typically interconvert on a fast time scale, methods for studying rapid tautomerism in the context of nucleic acids under biologically relevant aqueous conditions are also discussed.     

An RNA internal loop acts as a hinge to facilitate ribozyme folding and catalysis.

RNA molecules commonly consist of helical regions separated by internal loops, and in many cases these internal loops have been found to assume stable structures. We have examined the function and dynamics of an internal loop, J5/5a, that joins the two halves of the P4-P6 domain of the Tetrahymena self-splicing group I intron. P4-P6 RNAs with mutations in the J5/5a region showed nondenaturing gel electrophoretic mobilities and levels of Fe(II)-EDTA cleavage protection intermediate between those of wild-type RNA and a mutant incapable of folding into the native P4-P6 tertiary structure. Mutants with the least structured J5/5a loops behaved the most like wild-type P4-P6, and required smaller amounts of Mg2+ to rescue folding. The activity of reconstituted introns containing mutant P4-P6 RNAs correlated similarly with the nature of the J5/5a mutation. Our results suggest that, in solution, the P4-P6 RNA is in a two-state equilibrium between folded and unfolded states. We conclude that this internal loop mainly acts as a flexible hinge, allowing the coaxially stacked helical regions on either side of it to interact via specific tertiary contacts. To a lesser extent, the specific bases within the loop contribute to folding. Furthermore, it is crucial that the junction remain unstructured in the unfolded state. These conclusions cannot be derived from a simple examination of the P4-P6 crystal structure (Cate JH et al., 1996, Science 273:1678-1685), showing once again that structure determination must be supplemented with mutational and thermodynamic analysis to provide a complete picture of a folded macromolecule.     

Divalent metal ions influence catalysis and active-site accessibility in the cAMP-dependent protein kinase.

Phosphorylation of the peptide LRRASLG by the catalytic subunit of cAMP-dependent protein kinase was measured in the presence of various divalent metals to establish the role of electrophiles in the kinetic mechanism. Under conditions of low or high metal concentrations, the apparent second-order rate constant, kcat/Kpeptide, and the maximal rate constant, kcat, followed the trend Mg2+ > Co2+ > Mn2+. Competitive inhibition studies indicate that the former effect is not due to destabilization of the substrate complex, E.ATP.S. The effects of solvent viscosity on the steady-state kinetic parameters were interpreted according to a simple mechanism involving substrate binding, phosphotransfer, and product release steps and two metal chelation sites in the nucleotide pocket. Decreases in kcat and kcat/Kpeptide result mostly from attenuations in the dissociation rate constant for ADP and the association rate constant for the substrate, respectively. Decreases in the phosphoryl transfer rate constant have only negligible to moderate effects on these parameters. The low observed values for the association rate constant of the substrate indicate that the metals control the concentration of the productive binary form, Ea.ATP, and indirectly the accessibility of the active site. By comparison, Mg2+ is the best divalent metal catalyst because it uniformly lowers the transition state energies for all steps in the kinetic mechanism, permitting maximum flux of substrate to product. The data suggest that cAMP-dependent protein kinase uses metal ions to serve multiple roles in facilitating phosphotransfer and accelerating substrate association and product dissociation.     

Trigger-helix folding pathway and SI3 mediate catalysis and hairpin-stabilized pausing by Escherichia coli RNA polymerase

The conformational dynamics of the polymorphous trigger loop (TL) in RNA polymerase (RNAP) underlie multiple steps in the nucleotide addition cycle and diverse regulatory mechanisms. These mechanisms include nascent RNA hairpin-stabilized pausing, which inhibits TL folding into the trigger helices (TH) required for rapid nucleotide addition. The nascent RNA pause hairpin forms in the RNA exit channel and promotes opening of the RNAP clamp domain, which in turn stabilizes a partially folded, paused TL conformation that disfavors TH formation. We report that inhibiting TH unfolding with a disulfide crosslink slowed multiround nucleotide addition only modestly but eliminated hairpin-stabilized pausing. Conversely, a substitution that disrupts the TH folding pathway and uncouples establishment of key TH–NTP contacts from complete TH formation and clamp movement allowed rapid catalysis and eliminated hairpin-stabilized pausing. We also report that the active-site distal arm of the TH aids TL folding, but that a 188-aa insertion in the Escherichia coli TL (sequence insertion 3; SI3) disfavors TH formation and stimulates pausing. The effect of SI3 depends on the jaw domain, but not on downstream duplex DNA. Our results support the view that both SI3 and the pause hairpin modulate TL folding in a constrained pathway of intermediate states.     

RNA folding and unfolding

Single-molecule studies of RNA folding and unfolding are providing impressive details of the intermediates that occur and their rates of interconversion. The folding and unfolding of RNA are controlled by varying the concentration of magnesium ions and measuring fluorescence energy transfer, or by applying force to the RNA and measuring the end-to-end distance. The hierarchical nature of RNA folding - first secondary structure, then tertiary structure - makes the process susceptible to analysis and prediction.     

Metal ions in ribozyme folding and catalysis

Current research is reshaping basic theories regarding the roles of metal ions in ribozyme function. No longer viewed as strict metalloenzymes, some ribozymes can access alternative catalytic mechanisms depending on the identity and availability of metal ions. Similarly, reaction conditions can allow different folding pathways to predominate, with divalent cations sometimes playing opposing roles.