Defining the catalytic metal ion interactions in the Tetrahymena ribozyme reaction

Divalent metal ions play a crucial role in catalysis by many RNA and protein enzymes that carry out phosphoryl transfer reactions, and defining their interactions with substrates is critical for understanding the mechanism of biological phosphoryl transfer. Although a vast amount of structural work has identified metal ions bound at the active site of many phosphoryl transfer enzymes, the number of functional metal ions and the full complement of their catalytic interactions remain to be defined for any RNA or protein enzyme. Previously, thiophilic metal ion rescue and quantitative functional analyses identified the interactions of three active site metal ions with the 3'- and 2'-substrate atoms of the Tetrahymena group I ribozyme. We have now extended these approaches to probe the metal ion interactions with the nonbridging pro-S(P) oxygen of the reactive phosphoryl group. The results of this study combined with previous mechanistic work provide evidence for a novel assembly of catalytic interactions involving three active site metal ions. One metal ion coordinates the 3'-departing oxygen of the oligonucleotide substrate and the pro-S(P) oxygen of the reactive phosphoryl group; another metal ion coordinates the attacking 3'-oxygen of the guanosine nucleophile; a third metal ion bridges the 2'-hydroxyl of guanosine and the pro-S(P) oxygen of the reactive phosphoryl group. These results for the first time define a complete set of catalytic metal ion/substrate interactions for an RNA or protein enzyme catalyzing phosphoryl transfer.     

Synthesis and biochemical application of 2'-O-methyl-3'-thioguanosine as a probe to explore group I intron catalysis

Oligonucleotides containing 3'-S-phosphorothiolate linkages provide valuable analogues for exploring the catalytic mechanisms of enzymes and ribozymes, both to identify catalytic metal ions and to probe hydrogen-bonding interactions. Here, we have synthesized 2'-O-methyl-3'-thioguanosine to test a possible hydrogen-bonding interaction in the Tetrahymena ribozyme reaction. We developed an efficient method for the synthesis of 2'-O-methyl-3'-thioguanosine phosphoramidite in eight steps starting from 2'-O-methyl-N(2)-(isobutyryl) guanosine with 10.4% overall yield. Following incorporation into oligonucleotides using solid-phase synthesis, we used this new analogue to investigate whether the 3'-oxygen of the guanosine cofactor in the Tetrahymena ribozyme reaction serves as an acceptor for the hydrogen bond donated by the adjacent 2'-hydroxyl group. We show that regardless of whether the guanosine cofactor bears a 3'-oxygen or 3'-sulfur leaving group, replacing the adjacent 2'-hydroxyl group with a 2'-methoxy group incurs the same energetic penalty, providing evidence against an interaction. These results indicate that the hydrogen bond donated by the guanosine 2'-hydroxyl group contributes to catalytic function in a manner distinct from the U(-1) 2'-hydroxyl group.     

Metal ion coordination by the AGC triad in domain 5 contributes to group II intron catalysis

Group II introns require numerous divalent metal ions for folding and catalysis. However, because little information about individual metal ions exists, elucidating their ligands, functional roles and relationships to each other remains challenging. Here we provide evidence that an essential motif at the catalytic center of the group II intron, the AGC triad within domain 5 (D5), provides a ligand for a crucial metal ion. Sulfur substitution of the pro-Sp oxygen of the adenosine strongly disrupts D5 binding to a substrate consisting of an exon and domains 1-3 of the intron (exD123). Cd2+ rescues this effect by enabling the sulfur-modified D5 to bind to exD123 with wild type affinity and catalyze 5'-splice site cleavage. This switch in metal specificity implies that a metal ion interacts with D5 to mediate packing interactions with D123. This new D5 metal ion rescues the disruption of D5 binding and catalysis with a thermodynamic signature different from that of the metal ion that stabilizes the leaving group during the first step of splicing, suggesting the existence of two distinct metal ions.     

X-ray structure of the R69D phosphatidylinositol-specific phospholipase C enzyme: insight into the role of calcium and surrounding amino acids in active site geometry and catalysis

Phosphatidylinositol-specific phospholipase Cs (PLCs) are a family of phosphodiesterases that catalyze the cleavage of the P-O bond via transesterification using the internal hydroxyl group of the substrate as a nucleophile, generating the five-membered cyclic inositol phosphate as an intermediate or product. To better understand the role of calcium in the catalytic mechanism of PLCs, we have determined the X-ray crystal structure of an engineered PLC enzyme from Bacillus thuringiensis to 2.1 A resolution. The active site of this enzyme has been altered by substituting the catalytic arginine with an aspartate at position 69 (R69D). This single-amino acid substitution converted a metal-independent, low-molecular weight enzyme into a metal ion-dependent bacterial PLC with an active site architecture similar to that of the larger metal ion-dependent mammalian PLC. The Ca(2+) ion shows a distorted square planar geometry in the active site that allows for efficient substrate binding and transition state stabilization during catalysis. Additional changes in the positions of the catalytic general acid/general base (GA/GB) were also observed, indicating the interrelation of the intricate hydrogen bonding network involved in stabilizing the active site amino acids. The functional information provided by this X-ray structure now allows for a better understanding of the catalytic mechanism, including stereochemical effects and substrate interactions, which facilitates better inhibitor design and sheds light on the possibilities of understanding how protein evolution might have occurred across this enzyme family.     

Functional identification of ligands for a catalytic metal ion in group I introns

Many enzymes use metal ions within their active sites to achieve enormous rate acceleration. Understanding how metal ions mediate catalysis requires elucidation of metal ion interactions with both the enzyme and the substrate(s). The three-dimensional arrangement determined by X-ray crystallography provides a powerful starting point for identifying ground state interactions, but only functional studies can establish and interrogate transition state interactions. The Tetrahymena group I ribozyme is a paradigm for the study of RNA catalysis, and previous work using atomic mutagenesis and quantitative analysis of metal ion rescue behavior identified catalytic metal ions making five contacts with the substrate atoms. Here, we have combined atomic mutagenesis with site-specific phosphorothioate substitutions in the ribozyme backbone to establish transition state ligands on the ribozyme for one of the catalytic metal ions, referred to as M A. We identified the pro-S P oxygen atoms at nucleotides C208, A304, and A306 as ground state ligands for M A, verifying interactions suggested by the Azoarcus crystal structures. We further established that these interactions are present in the chemical transition state, a conclusion that requires functional studies, such as those carried out herein. Elucidating these active site connections is a crucial step toward an in-depth understanding of how specific structural features of the group I intron lead to catalysis.     

Crystal structure of a phage Twort group I ribozyme-product complex

Group I introns are catalytic RNAs capable of orchestrating two sequential phosphotransesterification reactions that result in self-splicing. To understand how the group I intron active site facilitates catalysis, we have solved the structure of an active ribozyme derived from the orf142-I2 intron from phage Twort bound to a four-nucleotide product RNA at a resolution of 3.6 A. In addition to the three conserved domains characteristic of all group I introns, the Twort ribozyme has peripheral insertions characteristic of phage introns. These elements form a ring that completely envelops the active site, where a snug pocket for guanosine is formed by a series of stacked base triples. The structure of the active site reveals three potential binding sites for catalytic metals, and invokes a role for the 2' hydroxyl of the guanosine substrate in organization of the active site for catalysis.     

Structure of the Tetrahymena ribozyme: base triple sandwich and metal ion at the active site

The Tetrahymena intron is an RNA catalyst, or ribozyme. As part of its self-splicing reaction, this ribozyme catalyzes phosphoryl transfer between guanosine and a substrate RNA strand. Here we report the refined crystal structure of an active Tetrahymena ribozyme in the absence of its RNA substrate at 3.8 A resolution. The 3'-terminal guanosine (omegaG), which serves as the attacking group for RNA cleavage, forms a coplanar base triple with the G264-C311 base pair, and this base triple is sandwiched by three other base triples. In addition, a metal ion is present in the active site, contacting or positioned close to the ribose of the omegaG and five phosphates. All of these phosphates have been shown to be important for catalysis. Therefore, we provide a picture of how the ribozyme active site positions both a catalytic metal ion and the nucleophilic guanosine for catalysis prior to binding its RNA substrate.     

Mechanistic inferences from the binding of ligands to LpxC, a metal-dependent deacetylase

The metal-dependent deacetylase LpxC catalyzes the first committed step of lipid A biosynthesis in Gram-negative bacteria. Accordingly, LpxC is an attractive target for the development of inhibitors that may serve as potential new antibiotics for the treatment of Gram-negative bacterial infections. Here, we report the 2.7 A resolution X-ray crystal structure of LpxC complexed with the substrate analogue inhibitor TU-514 and the 2.0 A resolution structure of LpxC complexed with imidazole. The X-ray crystal structure of LpxC complexed with TU-514 allows for a detailed examination of the coordination geometry of the catalytic zinc ion and other enzyme-inhibitor interactions in the active site. The hydroxamate group of TU-514 forms a bidentate chelate complex with the zinc ion and makes hydrogen bond interactions with conserved active site residues E78, H265, and T191. The inhibitor C-4 hydroxyl group makes direct hydrogen bond interactions with E197 and H58. Finally, the C-3 myristate moiety of the inhibitor binds in the hydrophobic tunnel of the active site. These intermolecular interactions provide a foundation for understanding structural aspects of enzyme-substrate and enzyme-inhibitor affinity. Comparison of the TU-514 complex with cacodylate and imidazole complexes suggests a possible substrate diphosphate binding site and highlights residues that may stabilize the tetrahedral intermediate and its flanking transition states in catalysis. Evidence of a catalytic zinc ion in the native zinc enzyme coordinated by H79, H238, D242, and two water molecules with square pyramidal geometry is also presented. These results suggest that the native state of this metallohydrolase may contain a pentacoordinate zinc ion, which contrasts with the native states of archetypical zinc hydrolases such as thermolysin and carboxypeptidase A.     

Modulation of individual steps in group I intron catalysis by a peripheral metal ion

Enzymes are complex macromolecules that catalyze chemical reactions at their active sites. Important information about catalytic interactions is commonly gathered by perturbation or mutation of active site residues that directly contact substrates. However, active sites are engaged in intricate networks of interactions within the overall structure of the macromolecule, and there is a growing body of evidence about the importance of peripheral interactions in the precise structural organization of the active site. Here, we use functional studies, in conjunction with published structural information, to determine the effect of perturbation of a peripheral metal ion binding site on catalysis in a well-characterized catalytic RNA, the Tetrahymena thermophila group I ribozyme. We perturbed the metal ion binding site by site-specifically introducing a phosphorothioate substitution in the ribozyme's backbone, replacing the native ligands (the pro-R (P) oxygen atoms at positions 307 and 308) with sulfur atoms. Our data reveal that these perturbations affect several reaction steps, including the chemical step, despite the absence of direct contacts of this metal ion with the atoms involved in the chemical transformation. As structural probing with hydroxyl radicals did not reveal significant change in the three-dimensional structure upon phosphorothioate substitution, the effects are likely transmitted through local, rather subtle conformational rearrangements. Addition of Cd(2+), a thiophilic metal ion, rescues some reaction steps but has deleterious effects on other steps. These results suggest that native interactions in the active site may have been aligned by the naturally occurring peripheral residues and interactions to optimize the overall catalytic cycle.     

Functional identification of catalytic metal ion binding sites within RNA

The viability of living systems depends inextricably on enzymes that catalyze phosphoryl transfer reactions. For many enzymes in this class, including several ribozymes, divalent metal ions serve as obligate cofactors. Understanding how metal ions mediate catalysis requires elucidation of metal ion interactions with both the enzyme and the substrate(s). In the Tetrahymena group I intron, previous work using atomic mutagenesis and quantitative analysis of metal ion rescue behavior identified three metal ions (MA, MB, and MC) that make five interactions with the ribozyme substrates in the reaction's transition state. Here, we combine substrate atomic mutagenesis with site-specific phosphorothioate substitutions in the ribozyme backbone to develop a powerful, general strategy for defining the ligands of catalytic metal ions within RNA. In applying this strategy to the Tetrahymena group I intron, we have identified the pro-SP phosphoryl oxygen at nucleotide C262 as a ribozyme ligand for MC. Our findings establish a direct connection between the ribozyme core and the functionally defined model of the chemical transition state, thereby extending the known set of transition-state interactions and providing information critical for the application of the recent group I intron crystallographic structures to the understanding of catalysis.     

Metal ion interaction with cosubstrate in self-splicing of group I introns.

The catalytic mechanism for self-splicing of the group I intron in the pre-mRNA from the nrdB gene in bacteriophage T4 has been investigated using 2'-amino- 2'-deoxyguanosine or guanosine as cosubstrates in the presence of Mg2+, Mn2+and Zn2+. The results show that a divalent metal ion interacts with the cosubstrate and thereby influences the efficiency of catalysis in the first step of splicing. This suggests the existence of a metal ion that catalyses the nucleophilic attack of the cosubstrate. Of particular significance is that the transesterification reactions of the first step of splicing with 2'-amino-2'-deoxyguanosine as cosubstrate are more efficient in mixtures containing either Mn2+or Zn2+together with Mg2+than with only magnesium ions present. The experiments in metal ion mixtures show that two (or more) metal ions are crucial for the self-splicing of group I introns and suggest the possibility that more than one of these have a direct catalytic role. A working model for a two-metal-ion mechanism in the transesterification steps is suggested.     

The pre-tRNA nucleotide base and 2'-hydroxyl at N(-1) contribute to fidelity in tRNA processing by RNase P

Fidelity in tRNA processing by the RNase P RNA from Escherichia coli depends, in part, on interactions with the nucleobase and 2' hydroxyl group of N(-1), the nucleotide immediately upstream of the site of RNA strand cleavage. Here, we report a series of biochemical and structure-function studies designed to address how these interactions contribute to cleavage site selection. We find that simultaneous disruption of cleavage site nucleobase and 2' hydroxyl interactions results in parallel reactions leading to correct cleavage and mis-cleavage one nucleotide upstream (5') of the correct site. Changes in Mg(2+) concentration and pH can influence the fraction of product that is incorrectly processed, with pH effects attributable to differences in the rate-limiting steps for the correct and mis-cleavage reaction pathways. Additionally, we provide evidence that interactions with the 2' hydroxyl group adjacent to the reactive phosphate group also contribute to catalysis at the mis-cleavage site. Finally, disruption of the adjacent 2'-hydroxyl contact has a greater effect on catalysis when pairing between the ribozyme and N(-1) is also disrupted, and the effects of simultaneously disrupting these contacts on binding are also non-additive. One implication of these results is that mis-cleavage will result from any combination of active site modifications that decrease the rate of correct cleavage beyond a certain threshold. Indeed, we find that inhibition of correct cleavage and corresponding mis-cleavage also results from disruption of any combination of active site contacts including metal ion interactions and conserved pairing interactions with the 3' RCCA sequence. Such redundancy in interactions needed for maintaining fidelity may reflect the necessity for multiple substrate recognition in vivo. These studies provide a framework for interpreting effects of substrate modifications on RNase P cleavage fidelity and provide evidence for interactions with the nucleobase and 2' hydroxyl group adjacent to the reactive phosphate group in the transition state.     

The role of magnesium ions and 2'-hydroxyl groups in the VS ribozyme-substrate interaction

The minimal substrate of the trans-cleaving Neurospora VS ribozyme has a stem-loop structure and interacts with the ribozyme by RNA tertiary interactions that remain only partially defined. The magnesium ion dependence of the catalytic parameters of a trans-cleaving VS-derived ribozyme were studied. The turnover number of the catalytic RNA was found to depend on the binding of at least three magnesium ions, with an apparent magnesium ion dissociation constant of 16mM, but K(M) was observed to be metal ion independent in the millimolar range. To address the role of 2'-hydroxyl groups of the VS substrate RNA in interactions with the ribozyme, 23 altered substrates, each with a single 2'-deoxyribonucleoside substitution, were synthesised and their kinetic properties in the VS ribozyme reaction were analysed. The removal of five 2'-hydroxyl groups, at positions G620, A621, U628, C629 and G630 inhibited the reaction, whereas at two sites, G623 and A639, reaction was stimulated by the modification. Substitution of G620 with a 2'-deoxynucleoside was expected to inhibit the reaction, in line with the critical role of this 2'-hydroxyl group in the transesterification reaction. Altered substrates in which a 2'-O-methyl nucleoside replaced A621, U628, C629 and G630 were prepared and characterised. Although removal of the hydroxyl group of A621 inhibited the turnover number of the ribozyme significantly, this activity was recovered upon 2'-O-methyl adenosine substitution, suggesting that the 2'-oxygen atom of this nucleoside forms an important contact within the ribozyme active site. A cluster of residues within the loop region of the substrate, were more modestly affected by 2'-deoxynucleoside substitution. In two cases, magnesium binding was impaired, suggesting that stem-loop I is a possible magnesium ion binding site.     

Catalytic mechanism of class B2 metallo-beta-lactamase

The initial nucleophilic substitution step of biapenem hydrolysis catalyzed by a subclass B2 metallo-beta-lactamase (CphA from Aeromonas hydrophila) is investigated using hybrid quantum mechanical/molecular mechanical methods and density functional theory. We focused on a recently proposed catalytic mechanism that involves a non-metal-binding water nucleophile in the active site of the monozinc CphA. Both theoretical models identified a single transition state featuring nearly concomitant nucleophilic addition and elimination steps, and the activation free energy from the potential of mean force calculations was estimated to be approximately 14 kcal/mol. The theoretical results also identified the general base for activating the water nucleophile to be the metal-binding Asp-120 rather than His-118, as suggested earlier. The protonation of Asp-120 leads to cleavage of the O(delta2)-Zn coordination bond, whereas the negatively charged nitrogen leaving group resulting from the ring opening replaces Asp-120 as the fourth ligand of the sole zinc ion. The electrophilic catalysis by the metal ion provides sufficient stabilization for the leaving group to avoid a tetrahedral intermediate. The theoretical studies provided detailed insights into the catalytic strategy of this unique metallo-beta-lactamase.     

Determination of the catalytic parameters of the N-terminal half of Escherichia coli ribonuclease E and the identification of critical functional groups in RNA substrates

Ribonuclease E is required for the rapid decay and correct processing of RNA in Escherichia coli. A detailed understanding of the hydrolysis of RNA by this and related enzymes will require the integration of structural and molecular data with quantitative measurements of RNA hydrolysis. Therefore, an assay for RNaseE that can be set up to have relatively high throughput while being sensitive and quantitative will be advantageous. Here we describe such an assay, which is based on the automated high pressure liquid chromatography analysis of fluorescently labeled RNA samples. We have used this assay to optimize reaction conditions, to determine for the first time the catalytic parameters for a polypeptide of RNaseE, and to investigate the RNaseE-catalyzed reaction through the modification of functional groups within an RNA substrate. We find that catalysis is dependent on both protonated and unprotonated functional groups and that the recognition of a guanosine sequence determinant that is upstream of the scissile bond appears to consist of interactions with the exocyclic 2-amino group, the 7N of the nucleobase and the imino proton or 6-keto group. Additionally, we find that a ribose-like sugar conformation is preferred in the 5'-nucleotide of the scissile phosphodiester bond and that a 2'-hydroxyl group proton is not essential. Steric bulk at the 2' position in the 5'-nucleotide appears to be inhibitory to the reaction. Combined, these observations establish a foundation for the functional interpretation of a three-dimensional structure of the catalytic domain of RNaseE when solved.     

Crystal structure of the leadzyme at 1.8 A resolution: metal ion binding and the implications for catalytic mechanism and allo site ion regulation

The leadzyme is a small ribozyme, derived from in vitro selection, which catalyzes site specific, Pb(2+)-dependent RNA cleavage. Pb(2+) is required for activity; Mg(2+) inhibits activity, while many divalent and trivalent ions enhance it. The leadzyme structure consists of an RNA duplex interrupted by a trinucleotide bulge. Here, crystal structures determined to 1.8 A resolution, both with Mg(2+) as the sole divalent counterion and with Mg(2+) and Sr(2+) (which mimics Pb(2+) with respect to binding but not catalysis), reveal the metal ion interactions with both the ground state and precatalytic conformations of the leadzyme. Mg(H(2)O)(6)(2+) ions bridge complementary strands of the duplex at multiple locations by binding tandem purines of one RNA strand in the major groove. At one site, Mg(H(2)O)(6)(2+) ligates the phosphodiester backbone of the trinucleotide bulge in the ground state conformation, but not in the precatalytic conformation, suggesting (a) Mg(2+) may inhibit leadzyme activity by stabilizing the ground state and (b) metal ions which displace Mg(2+) from this site may activate the leadzyme. Binding of Sr(2+) to the presumed catalytic Pb(2+) site in the precatalytic leadzyme induces local structural changes in a manner that would facilitate alignment of the catalytic ribose 2'-hydroxyl with the scissile bond for cleavage. These data support a model wherein binding of a catalytic ion to a precatalytic conformation of the leadzyme, in conjunction with the flexibility of the trinucleotide bulge, may facilitate structural rearrangements around the scissle phosphodiester bond favoring configurations that allow bond cleavage.     

Structure-function analysis from the outside in: long-range tertiary contacts in RNA exhibit distinct catalytic roles

The conserved catalytic core of the Tetrahymena group I ribozyme is encircled by peripheral elements. We have conducted a detailed structure-function study of the five long-range tertiary contacts that fasten these distal elements together. Mutational ablation of each of the tertiary contacts destabilizes the folded ribozyme, indicating a role of the peripheral elements in overall stability. Once folded, three of the five tertiary contact mutants exhibit defects in overall catalysis that range from 20- to 100-fold. These and the subsequent results indicate that the structural ring of peripheral elements does not act as a unitary element; rather, individual connections have distinct roles as further revealed by kinetic and thermodynamic dissection of the individual reaction steps. Ablation of P14 or the metal ion core/metal ion core receptor (MC/MCR) destabilizes docking of the substrate-containing P1 helix into tertiary interactions with the ribozyme's conserved core. In contrast, ablation of the L9/P5 contact weakens binding of the guanosine nucleophile by slowing its association, without affecting P1 docking. The P13 and tetraloop/tetraloop receptor (TL/TLR) mutations had little functional effect and small, local structural changes, as revealed by hydroxyl radical footprinting, whereas the P14, MC/MCR, and L9/P5 mutants show structural changes distal from the mutation site. These changes extended into regions of the catalytic core involved in docking or guanosine binding. Thus, distinct allosteric pathways couple the long-range tertiary contacts to functional sites within the conserved core. This modular functional specialization may represent a fundamental strategy in RNA structure-function interrelationships.     

Distinct sites of phosphorothioate substitution interfere with folding and splicing of the Anabaena group I intron

Although the active site of group I introns is phylogenetically conserved, subclasses of introns have evolved different mechanisms of stabilizing the catalytic core. Large introns contain weakly conserved 'peripheral' domains that buttress the core through predicted interhelical contacts, while smaller introns use loop-helix interactions for stability. In all cases, specific and non-specific magnesium ion binding accompanies folding into the active structure. Whether similar RNA-RNA and RNA-magnesium ion contacts play related functional roles in different introns is not clear, particularly since it can be difficult to distinguish interactions directly involved in catalysis from those important for RNA folding. Using phosphorothioate interference with RNA activity and structure in the small (249 nt) group I intron from Anabaena, we used two independent assays to detect backbone phosphates important for catalysis and those involved in intron folding. Comparison of the interference sites identified in each assay shows that positions affecting catalysis cluster primarily in the conserved core of the intron, consistent with conservation of functionally important phosphates, many of which are magnesium ion binding sites, in diverse group I introns, including those from Azoarcus and Tetrahymena. However, unique sites of folding interference located outside the catalytic core imply that different group I introns, even within the same subclass, use distinct sets of tertiary interactions to stabilize the structure of the catalytic core.     

Two distinct catalytic strategies in the hepatitis δ virus ribozyme cleavage reaction

The hepatitis delta virus (HDV) ribozyme and related RNAs are widely dispersed in nature. This RNA is a small nucleolytic ribozyme that self-cleaves to generate products with a 2',3'-cyclic phosphate and a free 5'-hydroxyl. Although small ribozymes are dependent on divalent metal ions under biologically relevant buffer conditions, they function in the absence of divalent metal ions at high ionic strengths. This characteristic suggests that a functional group within the covalent structure of small ribozymes is facilitating catalysis. Structural and mechanistic analyses have demonstrated that the HDV ribozyme active site contains a cytosine with a perturbed pK(a) that serves as a general acid to protonate the leaving group. The reaction of the HDV ribozyme in monovalent cations alone never approaches the velocity of the Mg(2+)-dependent reaction, and there is significant biochemical evidence that a Mg(2+) ion participates directly in catalysis. A recent crystal structure of the HDV ribozyme revealed that there is a metal binding pocket in the HDV ribozyme active site. Modeling of the cleavage site into the structure suggested that this metal ion can interact directly with the scissile phosphate and the nucleophile. In this manner, the Mg(2+) ion can serve as a Lewis acid, facilitating deprotonation of the nucleophile and stabilizing the conformation of the cleavage site for in-line attack of the nucleophile at the scissile phosphate. This catalytic strategy had previously been observed only in much larger ribozymes. Thus, in contrast to most large and small ribozymes, the HDV ribozyme uses two distinct catalytic strategies in its cleavage reaction.