Hammerhead Ribozyme Structure Research Articles

Structural Simplicity and Mechanistic Complexity in the Hammerhead Ribozyme.

Natural or full-length hammerhead ribozymes are up to 1000-fold more active than their minimal counterparts that lack a complex tertiary interaction that pre-organizes and stabilizes the ribozyme active site, positioning RNA functional groups to facilitate acid-base catalysis. The recent discovery that a single tertiary contact (an AU Hoogsteen pair) between Stems I and II confers essentially all of the enhanced activity greatly simplifies our understanding of the structural requirements for hammerhead ribozyme activity. In contrast, the simplest mechanistic interpretations are challenged with the presentation of more complex alternatives. These alternatives are elucidated and critically analyzed in the context of several of the active hammerhead ribozyme structures now available.

RNA Structure and Folding, edited by Dagmar Klostermeier and Christian Hammann. 2013. De Gruyter, Berlin/Boston.

Stephen A. Wainwright wrote in his book Axis and Circumference, The Cylindrical Shape of Plants and Animals a sentence, now frequently quoted, “Structure without function is a corpse, function without structure is a ghost.” With the advances and amazing progress in structural biology and bioinformatics, the descriptions and discussions about relationships or correlations linking structure to function, and vice versa, belong inevitably to almost any biological publication. However, as a colleague, Marc van Regenmortel, mentioned once, “Structure–function, infernal duo, it dissociates every microsecond!” For tackling this conundrum, biophysical methods and tools, coupled with the concepts of thermodynamics, are necessary to obtain a global understanding of biological and biochemical phenomena. The chapters in this book describe a whole gamut of techniques for assessing the biological reality and relevance of RNA structures and their molecular complexes. The large majority of our structural views on biological molecules comes from crystallography (celebrated this year since 2014 is the UNESCO International Year of Crystallography). However, we do see more frequently in the literature structures, surely beautiful and fascinating, but not necessarily biologically meaningful. RNA crystallography is probably highly prone to nonbiologically relevant structures or states because of multiple conformational states energetically accessible to RNA molecules. We need to remember only the striking saga of the hammerhead ribozyme structure, a prototypical example where excessively reductionist approaches by molecular biologists as well as structural biologists led the field astray for several years. Nowadays, we need to understand large complexes with several proteins and often several RNA strands. Proteins interact with each other in complex ways and, even without contacting the RNA component, require its presence for binding. The assembly of large ribonucleoparticles (RNPs) is therefore an interwoven and increasingly complex interplay between the protein and RNA parts. The biological relevance of the isolated complexes needs to be assessed regularly and at each step using a variety of techniques. Even after arduous and painstaking years of work, the crystallization and solution of the structure of an RNP complex demands that the biology it contains be ascertained. The book contains 15 chapters written by experts in the field. All of the chapters are presented in a common fashion with clear figures, several in color, and very readable formulae. The chapters can be grouped into five broad general methods. The first group covers the chemical approaches of RNA structure in solution: chemical probing (Mark Helm), bioorthogonal modifications (Claudia Hobartner et al.), and footprinting approaches (Melodie Duval et al.). The second group covers the main biophysical techniques: optical spectroscopy and calorimetry (by the editors), the elegant experiments tractable by gel electrophoresis (David Lilley), the rich possibilities of fluorescence energy transfer (Airat Gubaev and Dagmar Klostermeier), and the more recent single-molecule approaches using optical tweezers (Mathilde Bercy et al.). The third group covers the magnetic resonance techniques: nuclear magnetic resonance (Christina Mozes and Mirko Hennig) and pulsed electron paramagnetic resonance (Ivan Krstic et al.). The fourth major group covers the structural techniques of X-ray diffraction and cryo-electron microscopy. There is first a refreshing chapter on crystallization of RNA (Adrian Ferre-D'Amare). X-ray diffraction techniques as such are not described, but the use of X-ray diffraction coupled with atomic force microscopy (Alexander McPherson), small-angle X-ray scattering (Alexey Kikhney et al.), and cryo-electron microscopy (Jean-Francois Menetret et al.) illustrates the power and novelty that can be reached in this way. Finally, in the last group, two chapters cover some bioinformatic approaches: one for the prediction of secondary structure (Gerhard Steger and Robert Giegerich) and another for searching and annotating three-dimensional structures (Anton Petrov et al.). The book RNA Structure and Folding brings an up-to-date compilation of clear reviews with complete sets of references on most techniques regularly used to decipher the RNA structure and function. Students, researchers, and teachers should find it of great value.

Active-Site Monovalent Cations Revealed in a 1.55-Å-Resolution Hammerhead Ribozyme Structure

We have obtained a 1.55-Å crystal structure of a hammerhead ribozyme derived from Schistosoma mansoni under conditions that permit detailed observations of Na+ ion binding in the ribozyme's active site. At least two such Na+ ions are observed. The first Na+ ion binds to the N7 of G10.1 and the adjacent A9 phosphate in a manner identical with that previously observed for divalent cations. A second Na+ ion binds to the Hoogsteen face of G12, the general base in the hammerhead cleavage reaction, thereby potentially dissipating the negative charge of the catalytically active enolate form of the nucleotide base. A potential but more ambiguous third site bridges the A9 and scissile phosphates in a manner consistent with that of previous predictions. Hammerhead ribozymes have been observed to be active in the presence of high concentrations of monovalent cations, including Na+, but the mechanism by which monovalent cations substitute for divalent cations in hammerhead catalysis remains unclear. Our results enable us to suggest that Na+ directly and specifically substitutes for divalent cations in the hammerhead active site. The detailed geometry of the pre-catalytic active-site complex is also revealed with a new level of precision, thanks to the quality of the electron density maps obtained from what is currently the highest-resolution ribozyme structure in the Protein Data Bank.

Spiegelzymes: Sequence Specific Hydrolysis of L-RNA with Mirror Image Hammerhead Ribozymes and DNAzymes

In this manuscript we describe for the first time mirror image catalytic nucleic acids (Spiegelzymes), which hydrolyze sequence specifically L-ribonucleic acid molecules. The mirror image nucleic acid ribozymes designed are based upon the known hammerhead ribozyme and DNAzyme structures that contain L-ribose or L-deoxyribose instead of the naturally occurring D-ribose or D-deoxyribose, respectively. Both Spiegelzymes show similar hydrolytic activities with the same L-RNA target molecules and they also exhibit extra ordinary stabilities when tested with three different human sera. In this respect they are very similar to Spiegelmers (mirror image aptamers), which we had previously developed and for which it has been shown that they are non-toxic and non-immunogenic. Since we are also able to demonstrate that the hammerhead and DNAzyme Spiegelzymes can also hydrolyze mirror image oligonucleotide sequences, like they occur in Spiegelmers, in vivo, it seems reasonable to assume that Spiegelzymes may in principle be used as an antidote against Spiegelmers. Since the Spiegelzymes contain the same building blocks as the Spiegelmers, it can be expected that they will have similar favorable biological characteristics concerning toxicity and immunogenety. In trying to understand the mechanism of action of the Spiegelzymes described in this study, we have initiated for the first time a model building system with L-nucleic acids. The models for L-hammerhead ribozyme and L-DNAzyme interaction with the same L-RNA target will be presented.

Phylogenetic footprinting of non-coding RNA: hammerhead ribozyme sequences in a satellite DNA family of Dolichopodacave crickets (Orthoptera, Rhaphidophoridae)

BackgroundThe great variety in sequence, length, complexity, and abundance of satellite DNA has made it difficult to ascribe any function to this genome component. Recent studies have shown that satellite DNA can be transcribed and be involved in regulation of chromatin structure and gene expression. Some satellite DNAs, such as the pDo500 sequence family in Dolichopoda cave crickets, have a catalytic hammerhead (HH) ribozyme structure and activity embedded within each repeat.ResultsWe assessed the phylogenetic footprints of the HH ribozyme within the pDo500 sequences from 38 different populations representing 12 species of Dolichopoda. The HH region was significantly more conserved than the non-hammerhead (NHH) region of the pDo500 repeat. In addition, stems were more conserved than loops. In stems, several compensatory mutations were detected that maintain base pairing. The core region of the HH ribozyme was affected by very few nucleotide substitutions and the cleavage position was altered only once among 198 sequences. RNA folding of the HH sequences revealed that a potentially active HH ribozyme can be found in most of the Dolichopoda populations and species.ConclusionsThe phylogenetic footprints suggest that the HH region of the pDo500 sequence family is selected for function in Dolichopoda cave crickets. However, the functional role of HH ribozymes in eukaryotic organisms is unclear. The possible functions have been related to trans cleavage of an RNA target by a ribonucleoprotein and regulation of gene expression. Whether the HH ribozyme in Dolichopoda is involved in similar functions remains to be investigated. Future studies need to demonstrate how the observed nucleotide changes and evolutionary constraint have affected the catalytic efficiency of the hammerhead.

Capturing Hammerhead Ribozyme Structures in Action by Modulating General Base Catalysis

We have obtained precatalytic (enzyme–substrate complex) and postcatalytic (enzyme–product complex) crystal structures of an active full-length hammerhead RNA that cleaves in the crystal. Using the natural satellite tobacco ringspot virus hammerhead RNA sequence, the self-cleavage reaction was modulated by substituting the general base of the ribozyme, G12, with A12, a purine variant with a much lower pKa that does not significantly perturb the ribozyme's atomic structure. The active, but slowly cleaving, ribozyme thus permitted isolation of enzyme–substrate and enzyme–product complexes without modifying the nucleophile or leaving group of the cleavage reaction, nor any other aspect of the substrate. The predissociation enzyme-product complex structure reveals RNA and metal ion interactions potentially relevant to transition-state stabilization that are absent in precatalytic structures.

Capturing hammerhead ribozyme structures in action by modulating the rate of general base catalysis

Capturing hammerhead ribozyme structures in action by modulating the rate of general base catalysis

A discontinuous hammerhead ribozyme embedded in a mammalian messenger RNA

Structured RNAs embedded in the untranslated regions (UTRs) of messenger RNAs can regulate gene expression. In bacteria, control of a metabolite gene is mediated by the self-cleaving activity of a ribozyme embedded in its 5' UTR. This discovery has raised the question of whether gene-regulating ribozymes also exist in eukaryotic mRNAs. Here we show that highly active hammerhead ribozymes are present in the 3' UTRs of rodent C-type lectin type II (Clec2) genes. Using a hammerhead RNA motif search with relaxed delimitation of the non-conserved regions, we detected ribozyme sequences in which the invariant regions, in contrast to the previously identified continuous hammerheads, occur as two fragments separated by hundreds of nucleotides. Notably, a fragment pair can assemble to form an active hammerhead ribozyme structure between the translation termination and the polyadenylation signals within the 3' UTR. We demonstrate that this hammerhead structure can self-cleave both in vitro and in vivo, and is able to reduce protein expression in mouse cells. These results indicate that an unrecognized mechanism of post-transcriptional gene regulation involving association of discontinuous ribozyme sequences within an mRNA may be modulating the expression of several CLEC2 proteins that function in bone remodelling and the immune response of several mammals.

Effects of Various Promoter Derived Sequences on the Cleavage Kinetic of an Hammerhead Ribozyme Directed Against Cyclin E1 mRNA

Hammerhead ribozymes (HRz), catalytic RNA molecules capable of inducing the site-specific cleavage of a phosphodiester bond within an RNA molecule, are typically introduced into target cells by specific constructs (such as viral vectors) able to drive their expression from defined expression cassettes (promoter). This strategy implies the presence of promoter-derived sequences bound to the hammerhead ribozyme structure, a fact which can unpredictably affect HRz cleavage efficiency and eventually the biological effect. We explored the effects of promoter-derived sequences on the cleavage kinetics of an HRz targeted against a relevant cell cycle regulator, i.e. cyclin E1, implicated in the pathogenesis of several human diseases including in-stent restenosis and hepatocellular carcinoma. Sequences derived form the most commonly used promoters (CMV, T7, Pol I and Pol III promoters) were added to the minimal HRz structure and their effects on the cleavage kinetic constants k(cat) and K(m) evaluated in vitro under single turn-over conditions, using a mathematical model we recently developed. The different promoter derived sequences variably affected HRz cleavage efficiency (k(cat)/K(m)) with those derived from the pol III and from a truncated form of T7 promoter (T7-S), impairing maximally and minimally k(cat)/K(m), respectively. Additionally, the extra sequences tend to increase K(m) and to reduce k(cat). The extent of this effect depends both on the secondary RNA structure and on the length of the added sequences. In conclusion, these data, together with further work in cultured cells, can lead to the selection of optimal expression cassettes thus contributing to improve HRz efficacy, bringing these molecules closer to practical applications.

The Hammerhead Ribozyme Structure Brought in Line.

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The Hammerhead Ribozyme Structure Brought in Line

RNA catalysis seems to be considerably more wide spread than originally thought, with the most prominent example being the ribosome, where RNA catalyses the peptidyl-transferase reaction. Among the most and longest studied catalytic RNAs are the small nucleolytic ribozymes, such as the hairpin, VS, HDV and hammerhead ribozymes. They all catalyse the site-specific cleavage of their own phosphodiester backbone in cis or that of a substrate RNA in trans through a transesterification reaction involving the 2’-OH. A novel crystal structure of the hammerhead ribozyme has just been reported, and this should help to clarify a long-standing debate on the mechanism of catalysis. First identified in the 1980s as a catalytically active element in the replication cycle of certain viroids and the satellite RNA of plant viruses, the hammerhead ribozyme is the smallest naturally occurring RNA endonuclease. The motif has also been found in transcripts from the satellite DNA of amphibians, schistosomes, cave cricket and, most recently, encoded in the genomes of other eukaryotic organisms. The hammerhead ribozyme consists of a catalytic core of 11 conserved nucleotides that are flanked by three helices (Figure 1A). In the absence of divalent metal ions, the structure is extended, but upon addition of Mg , the RNA folds in two well-defined steps into a Y-shaped structure (Figure 1B), as deduced by Lilley and coworkers in studies using comparative gel electrophoresis, FRET, NMR and calorimetry. In this active conformation, a reversible transesterification reaction is catalysed by the hammerhead ribozyme (Scheme 1). 9] During cleavage, the 2’OH of nucleotide C17 is deprotonated and attacks the scissile 3’,5’ phosphodiester bond. Of the two cleavage products one carries a 2’,3’-cyclic phosphate, the other a 5’-hydroxy terminus. In the reverse (ligation) reaction, the 5’-oxygen attacks the cyclic phosphate. For the hammerhead ribozyme, however, the ligation does not proceed as efficiently as seen for the hairpin ribozyme. Both reactions proceed through the same, trigonal-bipyramidal pentacoordinated transition state (Scheme 1), thus meeting the principle of microscopic reversibility. This transition state was deduced from the observation that the chirality of the scissile phosphate, when exchanged for a phosphorothioate, was inverted during the course of the reaction, a hallmark of the SN2 mechanism. In the transition state, the 2’-OH of C17 has to be in line with the adjacent phosphorus and the 5’-oxygen of nucleotide 1.1 (Scheme 1). This requirement and other data detailed below gave rise to presumably the longest-standing debate in the ribozyme field. The first hammerhead ribozyme crystal structures showed a maximal deviation from the ACHTUNGTRENNUNGrequired in-line orientation of the three atoms, at 908. Hammerhead cleavage, however, could be achieved by soaking all RNA crystals with divalent metal ions. While the first observation argued for a ground-state structure to be present in the crystal, the second would indicate that no major rearrangements were necessary to reach the tran[a] R. Przybilski, Dr. C. Hammann AG Molecular Interactions Department of Genetics, University of Kassel Heinrich-Plett-Strasse 40 34132 Kassel (Germany) Fax: (+49)561-804-4800 E-mail : c.hammann@uni-kassel.de Figure 1. The hammerhead ribozyme. A) Secondary structure with stems I, II and III and the 11 conserved nucleotides (bold). Cleavage takes place between nucleotides 17 and 1.1, as indicated by an arrow. Numbers are given according to the conventional scheme. In minimal versions of the ribozyme, either stem I or II is closed by loops (dashed lines). B) Y-shaped conformation of the minimal version of the ribozyme upon addition of magnesium. Naturally occurring ribozymes are endowed with tertiary stabilising structures formed between C) loops L1 and L2 or D) loop L1 and bulge B2 in stem II. The ribozyme format shown in (D) was used for crystallisation.

Viroids: unusual small pathogenic RNAs.

Viroids are small (about 300 nucleotides), single-stranded, circular, non-encapsidated pathogenic RNA molecules. They do not code for proteins and thus depend on plant host enzymes for their replication and other functions. They induce plant diseases by direct interaction with host factors but the mechanism of pathogenicity is still unknown. They can alter the expression of selected plant genes important for growth and development. Viroids belong to two families, the Avsunviroidae and the Pospiviroidae. Viroids of the Avsunviroidae family adopt a branched or quasi rod-like secondary structure in their native state. Members of the Pospiviroidae family adopt a rod-like secondary structure. In such native structures five structural/functional domains have been identified: central (C), pathogenicity, variable and two terminal domains. The central conserved region (CCR) within the C domain characterizes viroids of the Pospiviroidae. Specific secondary structures of this region play an important role in viroid replication and processing. Viroids of the Avsunviroidae family lack a CCR but possess self-cleaving properties by forming hammerhead ribozyme structures; they accumulate and replicate in chloroplasts, whereas members of the Pospiviroidae family have a nuclear localization. Viroid replication occurs via a rolling circle mechanism using either a symmetric or asymmetric pathway in three steps, RNA transcription, processing and ligation.

Viroids: unusual small pathogenic RNAs.

Viroids are small (about 300 nucleotides), single-stranded, circular, non-encapsidated pathogenic RNA molecules. They do not code for proteins and thus depend on plant host enzymes for their replication and other functions. They induce plant diseases by direct interaction with host factors but the mechanism of pathogenicity is still unknown. They can alter the expression of selected plant genes important for growth and development. Viroids belong to two families, the Avsunviroidae and the Pospiviroidae. Viroids of the Avsunviroidae family adopt a branched or quasi rod-like secondary structure in their native state. Members of the Pospiviroidae family adopt a rod-like secondary structure. In such native structures five structural/functional domains have been identified: central (C), pathogenicity, variable and two terminal domains. The central conserved region (CCR) within the C domain characterizes viroids of the Pospiviroidae. Specific secondary structures of this region play an important role in viroid replication and processing. Viroids of the Avsunviroidae family lack a CCR but possess self-cleaving properties by forming hammerhead ribozyme structures; they accumulate and replicate in chloroplasts, whereas members of the Pospiviroidae family have a nuclear localization. Viroid replication occurs via a rolling circle mechanism using either a symmetric or asymmetric pathway in three steps, RNA transcription, processing and ligation.

A pH-dependent conformational change, rather than the chemical step, appears to be rate-limiting in the hammerhead ribozyme cleavage reaction

We have investigated the chemical basis for a previously observed 7.8 Å conformational change in the hammerhead ribozyme that positions the substrate for in-line attack. We have found that the conformational change can only be observed at or above pH 8.5 (in the presence of Co2+) and requires the presence of an ionizable 2′-OH at the cleavage site, and note that this observed apparent pKa of 8.5 for the conformational change is within experimental error (±0.5) of the previously reported apparent kinetic pKa of 8.5 for the hammerhead ribozyme in the presence of Co2+. We have solved two crystal structures of hammerhead ribozymes having 2′-OCH3 or 2′-F substitutions at the cleavage site and have found that these will not undergo a conformational change equivalent to that observed for the hammerhead ribozyme having an unmodified attacking nucleophile under otherwise identical conditions. We have also characterized the kinetics of cleavage in the crystal. In addition to verifying that the particular sequence of RNA that we crystallized cleaves faster in the crystal than in solution, we also find that the extent of cleavage in the crystal is complete, unlike in solution where this and most other hammerhead ribozyme substrates are cleaved only to about 70 % completion. The initial cleavage rate in the crystal obeys the expected log-linear relation between cleavage-rate and pH with a slope of 0.7, as has been observed for other hammerhead ribozyme sequences in solution, indicating that in both the crystal and in solution the pH-dependent step is rate-limiting. However, the cleavage rate in the crystal is biphasic, with the most dramatic distinction between initial (slower) and final (faster) phases appearing at pH 6.0. The initial phase corresponds to the pH-dependent cleavage rate observed in solution, but the second, faster phase is roughly pH-independent and closely parallels the cleavage rate observed at pH 8 (0.4/minute). This result is particularly remarkable because it entails that the rapidly cleaving phase at pH 6 is comparable to the cleavage rate for the fastest cleaving hammerhead ribozymes at pH 6. Based upon these observations, we conclude that the pH-dependent conformational change is the rate-determining step under standard conditions for the hammerhead ribozyme self-cleavage reaction, and that an ionizable 2′-proton at cleavage site is required for this conformational change. We further hypothesize that deprotonation of the cleavage-site 2′-oxygen drives this conformational change.

Hammerhead ribozyme structure and function in plant RNA replication

Hammerhead ribozyme structure and function in plant RNA replication

Corrigendum: Does a Single Metal Ion Bridge the A-9 and Scissile Phosphate Groups in the Catalytically Active Hammerhead Ribozyme Structure?

2a2 is the most commonly rearranged gene in the human Vλ locus. It has been postulated that certain immunoglobulin genes (including 2a2) are rearranged preferentially because their germline sequences encode structures capable of binding to a range of antigens. Somatic mutation could then increase the specificity and affinity of binding to a particular antigen.We studied the properties of five IgG molecules in which the same heavy chain was paired with different light chains derived from 2a2. The pattern of somatic mutations in 2a2 was shown to be crucial in conferring the ability to bind DNA, but two different patterns of mutation each conferred this ability.Computer-generated models of the three-dimensional structures of these antibodies illustrate the ability of 2a2 to form a DNA binding site in different ways. Somatic mutations at the periphery of the DNA binding site were particularly important. In two different light chains, mutations to arginine at different sites in the complementarity determining regions (CDRs) enhanced binding to DNA. In a third light chain, however, mutation to arginine at a different site blocked binding to DNA.

Hammerhead-mediated processing of satellite pDo500 family transcripts from Dolichopoda cave crickets.

This work reports the discovery and functional characterization of catalytically active hammerhead motifs within satellite DNA of the pDo500 family from several DOLICHOPODA: cave cricket species. We show that in vitro transcribed RNA of some members of this satellite DNA family do self-cleave in vitro. This self-cleavage activity is correlated with the efficient in vivo processing of long primary transcripts into monomer-sized RNA. The high sequence conservation of the satellite pDo500 DNA family among genetically isolated DOLICHOPODA: schiavazzii populations, as well as other DOLICHOPODA: species, along with the fact that satellite members are actively transcribed in vivo suggests that the hammerhead-encoding satellite transcripts are under selective pressure, perhaps because they fulfil an important physiological role or function. Remarkably, this is the third example of hammerhead ribozyme structures associated with transcribed repetitive DNA sequences from animals. The possibility that such an association may not be purely coincidental is discussed.

Does a single metal ion bridge the A-9 and scissile phosphate groups in the catalytically active hammerhead ribozyme structure?

We have constructed a model structure that we believe represents the strongest possible physically and chemically reasonable representation of a hypothesized catalytically active hammerhead ribozyme structure in which a single divalent metal ion bridges the A9 and scissile phosphate groups. It has been proposed that such a structure arises from a conformational change in which the so-called ground-state structure (as observed by X-ray crystallography) rearranges in such a way that the pro-R oxygen atoms of both the A9 and scissile phosphate groups are directly coordinated by a single divalent metal ion in the transition-state of the hammerhead ribozyme cleavage reaction. We show that even the small subset of possible model structures that are consistent with these requirements, and that are stereochemically and sterically reasonable, are contradicted by experimental evidence. We also demonstrate that even a minimal subset of assumptions, i.e. that stems I and II are helical and that the two phosphate groups are coordinated by a divalent metal ion in the standard octahedral geometry, are sufficient to lead to this contradiction. We therefore conclude that such a mechanism of hammerhead ribozyme catalysis is untenable, at least in its present formulation.

The Mechanism of Phosphodiester Hydrolysis: Near In-line Attack Conformations in the Hammerhead Ribozyme

The hammerhead ribozyme is a small RNA molecule capable of self-cleavage at a specific site in the phosphodiester backbone. The mechanism of hydrolysis involves in-line nucleophilic attack by the 2‘-hydroxyl of residue 17 on the adjoining phosphorus of residue 1.1, resulting in the formation of a 2‘,3‘-cyclic phosphate ester on residue 17 (C17) and elimination of the 5‘-hydroxyl group of residue 1.1 (A1.1). Unconstrained molecular dynamics (MD) simulations on the recently solved crystallographic unmodified hammerhead ribozyme structure were performed in solution using two crystallographic Mg2+ ions. The simulations indicate that near in-line attack conformations (NACs), in which the distance of the 2‘-oxygen of C17 to the phosphorus of A1.1 is ≤3.25 Å and the C17 2‘O−A1.1 P−A1.1 O5‘ angle of displacement is ≥150°, form approximately 18% of the simulation time. The motions leading to these catalytically competent conformations are discussed. Stems I and II of the hammerhead ribozyme structure, released from the pseudo-continuous helix and other crystallographic constraints, move toward each other. This, along with the Mg2+ ion bound at the pro-R phosphate oxygen of residue 1.1, prompts torsional rotations in the phosphodiester backbone primarily near the active site. These rotations lead to the unstacking of residues C17 and G5 from A6. Residue G5 then interacts with other conserved residues in the structure and does not stack with A6 again. Following spontaneous backbone conformational rearrangements, a ribose sugar pucker flip from C3‘-endo to C2‘-endo occurs in the nucleotide containing the 2‘-hydroxyl nucleophile. The base of residue C17 then restacks with the base of residue A6, and NACs occur shortly thereafter. During the simulations, one Mg2+ ion remains coordinated to the pro-R phosphate oxygen of the C17 nucleotide, while the other Mg2+ ion serves a structural role and does not participate in the transesterification reaction. The formation of NACs is spontaneous during the simulation and in a replicate simulation.

Entropically Driven Enhancement of Cleavage Activity of a DNA-Armed Hammerhead Ribozyme: Mechanism of Action of Hammerhead Ribozymes

Recent X-ray structures of hammerhead ribozymes demanded rearrangement of the scissile phosphate backbone into a conformation suitable for in-line attack by the 2‘-oxygen in the transition state or prior to it. As part of our efforts to detect the putative conformational change and also to characterize the transition state structure of a hammerhead ribozyme-catalyzed reaction, kinetic and thermodynamic parameters of a chimeric RNA/DNA ribozyme were compared with the corresponding values of an all-RNA ribozyme. Kinetic analysis revealed that (i) DNA portions enhanced kcat in the entire pH region between 6 and 9, and at all temperatures between 0 and 60 °C; (ii) the origin of the acceleration in kcat by the chimeric DNA-armed ribozyme was accomplished by two distinctly different phenomena: at the temperatures below 25 °C, the enhancement originated from the acceleration of dissociation of the products from the reaction complex (kdiss), whereas at temperatures 25−45 °C, the DNA-portions enhanced the chemica...