Terbium-mediated footprinting probes a catalytic conformational switch in the antigenomic hepatitis delta virus ribozyme

The two forms of the hepatitis delta virus ribozyme are derived from the genomic and antigenomic RNA strands of the human hepatitis delta virus (HDV), where they serve a crucial role in pathogen replication by catalyzing site-specific self-cleavage reactions. The HDV ribozyme requires divalent metal ions for formation of its tertiary structure, consisting of a tight double-nested pseudoknot, and for efficient self- (or cis-) cleavage. Comparison of recently solved crystal structures of the cleavage precursor and 3' product indicates that a significant conformational switch is required for catalysis by the genomic HDV ribozyme. Here, we have used the lanthanide metal ion terbium(III) to footprint the precursor and product solution structures of the cis-acting antigenomic HDV ribozyme. Inhibitory Tb(3+) binds with high affinity to similar sites on RNA as Mg(2+) and subsequently promotes slow backbone scission. We find subtle, yet significant differences in the terbium(III) footprinting pattern between the precursor and product forms of the antigenomic HDV ribozyme, consistent with differences in conformation as observed in the crystal structures of the genomic ribozyme. In addition, UV melting profiles provide evidence for a less tight tertiary structure in the precursor. In both the precursor and product we observe high-affinity terbium(III) binding sites in joining sequence J4/2 (Tb(1/2) approximately 4 microM) and loop L3, which are key structural components forming the catalytic core of the HDV ribozyme, as well as in several single-stranded regions such as J1/2 and the L4 tetraloop (Tb(1/2) approximately 50 microM). Sensitized luminescence spectroscopy confirms that there are at least two affinity classes of Tb(3+) binding sites. Our results thus demonstrate that a significant conformational change accompanies catalysis in the antigenomic HDV ribozyme in solution, similar to the catalytic conformational switch observed in crystals of the genomic form, and that structural and perhaps catalytic metal ions bind close to the catalytic core.     

Catalytic core structure of the trans-acting HDV ribozyme is subtly influenced by sequence variation outside the core

The human pathogenic hepatitis delta virus (HDV) employs a unique self-cleaving catalytic RNA motif, the HDV ribozyme, during double-rolling circle replication. Fluorescence spectroscopy, circular dichroism, terbium(III) footprinting, and X-ray crystallography of precursor and product forms have revealed that a conformational change accompanies catalysis. In addition, fluorescence resonance energy transfer (FRET) has previously been used on a trans-acting HDV ribozyme to demonstrate surprisingly significant catalytic and global conformational effects of substrate analogues with varying 5' sequences, which reside as dangling overhangs outside the catalytic core. Here, we use the fluorescent guanine analogue 2-aminopurine (AP) in nucleotide position 76, immediately downstream of the catalytically involved C75, to monitor the relative structural effects of these substrate analogues on the ribozyme's trefoil turn of the catalytic core. Steady-state and time-resolved AP fluorescence spectroscopies show that the binding of each substrate analogue induces a unique local conformation with a specific AP76 stacking equilibrium. Binding of the 3' product results in a relative increase in AP fluorescence, suggesting that AP76 becomes more unstacked upon catalysis. These local conformational changes are kinetically concomitant with global conformational changes monitored by FRET. Finally, the rate constant of the local conformational change upon 3' product binding is fast and independent of 3' product concentration yet Mg2+ dependent. Our results demonstrate that the trefoil turn of the HDV ribozyme catalytic core is in a state of dynamic equilibrium not captured by static crystal structures and is highly sensitive to the identity of the 5' sequence and Mg2+ ions.     

Reaction pathway of the trans-acting hepatitis delta virus ribozyme: a conformational change accompanies catalysis.

The hepatitis delta virus (HDV), an infectious human pathogen and satellite of hepatitis B virus, leads to intensified disease symptoms, including progression to liver cirrhosis. Both the circular RNA genome of HDV and its complementary antigenome contain the same cis-cleaving catalytic RNA motif that plays a crucial role in virus replication. Previously, the high-resolution crystal structure of the product form of a cis-acting genomic HDV ribozyme has been determined, while a trans-acting version of the ribozyme was used to dissect the cleavage reaction pathway. Using fluorescence resonance energy transfer (FRET) on a synthetic trans-cleaving form of the ribozyme, we are able to directly observe substrate binding (at a rate constant k(on) of 7.8 x 10(6) M(-1) min(-1) at pH 7.5, 11 mM MgCl(2), and 25 degrees C) and dissociation (at 0.34 min(-1)). Steady-state and time-resolved FRET experiments in solution and in nondenaturing gels reveal that the substrate (precursor) complex is slightly more compact (by approximately 3 A) than the free ribozyme, yet becomes significantly extended (by approximately 15 A) upon cleavage and product complex formation. We also find that trans cleavage is characterized by a high transition-state entropy (-26 eu). We propose that the significant global conformational change that we observe between the precursor and product structures occurs on the reaction trajectory into a constrained product complex-like transition state. Our observations may present the structural basis of the recently described utilization of intrinsic substrate binding energy to the overall catalytic rate enhancement by the trans-acting HDV ribozyme.     

Magnesium dependence of the amplified conformational switch in the trans-acting hepatitis delta virus ribozyme

The ability of divalent metal ions to participate in both structure formation and catalytic chemistry of RNA enzymes (ribozymes) has made it difficult to separate their cause and effect in ribozyme function. For example, the recently solved crystal structures of precursor and product forms of the cis-cleaving genomic hepatitis delta virus (HDV) ribozyme show a divalent metal ion bound in the active site that is released upon catalysis due to an RNA conformational change. This conformational switch is associated with a repositioning of the catalytically involved base C75 in the active-site cleft, thus controlling catalysis. These findings confirm previous data from fluorescence resonance energy transfer (FRET) on a trans-acting form of the HDV ribozyme that found a global conformational change to accompany catalysis. Here, we further test the conformational switch model by measuring the Mg(2+) dependence of the global conformational change of the trans-acting HDV ribozyme, using circular dichroism and time-resolved FRET as complementary probes of secondary and tertiary structure formation, respectively. We observe significant differences in both structure and Mg(2+) affinity of the precursor and product forms, in the presence and absence of 300 mM Na(+) background. The precursor shortens while the product extends with increasing Mg(2+) concentration, essentially amplifying the structural differences observed in the crystal structures. In addition, the precursor has an approximately 2-fold and approximately 13-fold lower Mg(2+) affinity than the product in secondary and tertiary structure formation, respectively. We also have compared the C75 wild-type with the catalytically inactive C75U mutant and find significant differences in global structure and Mg(2+) affinity for both their precursor and product forms. Significantly, the Mg(2+) affinity of the C75 wild-type is 1.7-2.1-fold lower than that of the C75U mutant, in accord with the notion that C75 is essential for a catalytic conformational change that leads to a decrease in the local divalent metal ion affinity and release of a catalytic metal. Thus, a consistent picture emerges in which divalent metal ions and RNA functional groups are intimately intertwined in affecting structural dynamics and catalysis in the HDV ribozyme.     

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.     

Trans-acting hepatitis delta virus ribozyme: catalytic core and global structure are dependent on the 5' substrate sequence

The hepatitis delta virus (HDV), an infectious human pathogen affecting millions of people worldwide, leads to intensified disease symptoms, including progression to liver cirrhosis upon coinfection with its helper virus, HBV. Both the circular RNA genome of HDV and its complementary antigenome contain a common cis-cleaving catalytic RNA motif, the HDV ribozyme, which plays a crucial role in viral replication. Previously, the crystal structure of the product form of the cis-acting genomic HDV ribozyme has been determined, and the precursor form has been suggested to be structurally similar. In contrast, solution studies by fluorescence resonance energy transfer (FRET) on a trans-cleaving form of the ribozyme have shown significant global conformational changes upon catalysis, while 2-aminopurine (AP) fluorescence assays have detected concomitant local conformational changes in the catalytic core. Here, we augment these studies by using terbium(III) to probe the structure of the trans-acting HDV ribozyme at nucleotide resolution. We observe significant structural differences between the precursor and product forms, especially in the P1.1 helix and the trefoil turn in the single-stranded region connecting P4 and P2 (termed J4/2) of the catalytic core. We show, using terbium(III) footprinting and sensitized luminescence spectroscopy as well as steady-state, time-resolved, and gel-mobility FRET assays on a systematic set of substrates, that the substrate sequence immediately 5' to the cleavage site significantly modulates these local as well as resultant global structural differences. Our results suggest a structural basis for the previously observed impact of the 5' substrate sequence on catalytic activity.     

Local conformational changes in the catalytic core of the trans-acting hepatitis delta virus ribozyme accompany catalysis

The hepatitis delta virus (HDV) is a human pathogen and satellite RNA of the hepatitis B virus. It utilizes a self-cleaving catalytic RNA motif to process multimeric intermediates in the double-rolling circle replication of its genome. Previous kinetic analyses have suggested that a particular cytosine residue (C(75)) with a pK(a) close to neutrality acts as a general acid or base in cleavage chemistry. The crystal structure of the product form of a cis-acting HDV ribozyme shows this residue positioned close to the 5'-OH leaving group of the reaction by a trefoil turn in the RNA backbone. By modifying G(76) of the trefoil turn of a synthetic trans-cleaving HDV ribozyme to the fluorescent 2-aminopurine (AP), we can directly monitor local conformational changes in the catalytic core. In the ribozyme-substrate complex (precursor), AP fluorescence is strongly quenched, suggesting that AP(76) is stacked with other bases and that the trefoil turn is not formed. In contrast, formation of the product complex upon substrate cleavage or direct product binding results in a significant increase in fluorescence, consistent with AP(76) becoming unstacked and solvent-exposed as evidenced in the trefoil turn. Using AP fluorescence and fluorescence resonance energy transfer (FRET) in concert, we demonstrate that this local conformational change in the trefoil turn is kinetically coincidental with a previously observed global structural change of the ribozyme. Our data show that, at least in the trans-acting HDV ribozyme, C(75) becomes positioned for reaction chemistry only along the trajectory from precursor to product.     

Structural roles of monovalent cations in the HDV ribozyme

The hepatitis delta virus (HDV) ribozyme catalyzes viral RNA self-cleavage through general acid-base chemistry in which an active-site cytidine and at least one metal ion are involved. Monovalent metal ions support slow catalysis and were proposed to substitute for structural, but not catalytic, divalent metal ions in the RNA. To investigate the role of monovalent cations in ribozyme structure and function, we determined the crystal structure of the precursor HDV ribozyme in the presence of thallium ions (Tl(+)). Two Tl(+) ions can occupy a previously observed divalent metal ion hexahydrate-binding site located near the scissile phosphate, but are easily competed away by cobalt hexammine, a magnesium hexahydrate mimic and potent reaction inhibitor. Intriguingly, a third Tl(+) ion forms direct inner-sphere contacts with the ribose 2'-OH nucleophile and the pro-S(p) scissile phosphate oxygen. We discuss possible structural and catalytic implications of monovalent cation binding for the HDV ribozyme mechanism.     

Interaction between RNA molecules of a two-component trans analog of antigenomic HDV ribozyme

A study was made of the association of the RNA components forming a B:LS two-component rans analog of the antigenomic HDV ribozyme. The B:LS ribozyme differed from known trans ribozymes in the sizes and nucleotide sequences of its components (33 and 34 nt, respectively), the topology of its functional parts, and the lack of a very short cleavage product. Compared to the cis ribozyme, B:LS showed similar dependences on the reaction conditions (Mg²⁺ concentration, pH, temperature) and a similar biphasic kinetic curve of self-cleavage. The kinetic model of B:LS self-cleavage (available at www.cardio.ru/labgen/RZ_e.html) describes a possible cause of the biphasic kinetic curve as a change in the rate-limiting step of consecutive conformational transitions accompanying self-cleavage. Another possible cause is an interaction between the molecules involved in cleavage, i.e., multimerization of whole ribozyme molecules with their components or the reaction products. B:LS provides a convenient model for studying such interactions, since the mode of component binding allows generation of 1B:2LS and 2B:1LS complexes as well as complexes with the cleavage products. Nondenaturing PAGE was used to study the factors affecting association and dissociation of the ribozyme components. The possibility of interactions between the RNA components of the cis and trans ribozymes was demonstrated experimentally. It was shown that the ribozyme is capable of multimerization when LS is in excess over B and that the cleavage products are not significantly involved in this process. The results suggest intermolecular interactions for the cleavage of the natural cis ribozyme.     

Formation of the P1.1 pseudoknot is critical for both the cleavage activity and substrate specificity of an antigenomic trans-acting hepatitis delta ribozyme

Hepatitis delta virus RNAs possess self-cleavage activities that produce 2′,3′-cyclic phosphate and 5′-hydroxyl termini (i.e. cis-acting delta ribozyme). Trans-acting delta ribozymes have been engineered by removing a junction from the cis version, thereby producing one molecule possessing the substrate sequence and the other the catalytic domain. According to the pseudoknot model, the secondary structure of the delta ribozyme includes a pseudoknot (i.e. P1.1 stem) formed by two base pairs from residues of the L3 loop and J1/4 junction. A collection of 48 P1.1 stem mutants was synthesized in order to provide an original characterization of both the importance and the structure of this pseudoknot in a trans-acting version of the ribozyme. Several structural differences were noted compared to the results reported for cis-acting ribozymes. For example, a combination of two stable Watson–Crick base pairs composing the essential P1.1 stem was demonstrated to be crucial for a significant level of activity, while the cis version required only one base pair. In addition, we present the first physical evidences revealing that the composition of the P1.1 stem affects the substrate specificity for ribozyme cleavage. Depending on the residues forming the J1/4 junction, non-productive ribozyme–substrate complexes can be observed. This phenomenon is proposed to be important for further development of a gene-inactivation system based on delta ribozyme.     

Properties of Antigenomic HDV Ribozyme Consisting of Two RNA Chains

B:LS ribozyme, a trans-variant of naturally occurring HDV ribozyme, has been constructed. The ribozyme consists of a substrate-containing LS chain and a catalytic B chain and differs from previously constructed trans-ribozymes in the length and nucleotide sequence of its oligonucleotide chains (33 and 34 bp, respectively). The chains readily associate with each other at room temperature, at which the LS cleavage reaction is negligible, which makes it possible to investigate association of the intact chains. At the same time, the self-cleavage rate constant for the trans-ribozyme B:LS at 50°C is close to those for the previously studied permuted cis-ribozymes, especially the LSB variant. In addition, the dependence of trans-ribozyme on reaction conditions (Mg²⁺ concentration, pH, and temperature) resembled that of cis-ribozyme. Similar to other trans-ribozymes, B:LS ribozyme demonstrated the ability for multiple turnover of the B strand with an excess of the substrate LS chain. The kinetic model of the self-cleavage reaction for B:LS is presented at http://www.cardio.ru/labgen/RZ_r.html. Taken together, our results show that the novel trans-variant of HDV ribozyme can be used as a model for analyzing the process of HDV ribozyme self-cleavage.     

The genomic HDV ribozyme utilizes a previously unnoticed U-turn motif to accomplish fast site-specific catalysis

The genome of the human hepatitis delta virus (HDV) harbors a self-cleaving catalytic RNA motif, the genomic HDV ribozyme, whose crystal structure shows the dangling nucleotides 5′ of the cleavage site projecting away from the catalytic core. This 5′-sequence contains a clinically conserved U − 1 that we find to be essential for fast cleavage, as the order of activity follows U − 1 > C − 1 > A − 1 > G − 1, with a >25-fold activity loss from U − 1 to G − 1. Terbium(III) footprinting detects conformations for the P1.1 stem, the cleavage site wobble pair and the A-minor motif of the catalytic trefoil turn that depend on the identity of the N − 1 base. The most tightly folded catalytic core, resembling that of the reaction product, is found in the U − 1 wild-type precursor. Molecular dynamics simulations demonstrate that a U − 1 forms the most robust kink around the scissile phosphate, exposing it to the catalytic C75 in a previously unnoticed U-turn motif found also, for example, in the hammerhead ribozyme and tRNAs. Strikingly, we find that the common structural U-turn motif serves distinct functions in the HDV and hammerhead ribozymes.     

Coordinated control of a designed trans-acting ligase ribozyme by a loop-receptor interaction

We previously developed a synthetic cis-acting RNA ligase ribozyme with 3′-5′ joining activity termed “DSL” (designed and selected ligase). DSL was easily transformed into a trans-acting form because of its highly modular architecture. In this study, we investigated the modular properties and turnover capabilities of a trans-acting DSL, tDSL-1/GUAA. tDSL-1/GUAA exhibited remarkably high activity compared with the parental cis-acting DSL, and it attained a high turnover number. Taken together, the results indicate that a loop-receptor interaction plays a significant role in determining the activity of the trans-acting ribozyme and in its ability to perform multiple turnovers of the reaction.     

The role of phosphate groups in the VS ribozyme-substrate interaction

The VS ribozyme trans-cleavage substrate interacts with the catalytic RNA via tertiary interactions. To study the role of phosphate groups in the ribozyme–substrate interaction, 18 modified substrates were synthesized, where an epimeric phosphorothioate replaces one of the phosphate diester linkages. Sites in the stem–loop substrate where phosphorothioate substitution impaired reaction cluster in two regions. The first site is the scissile phosphate diester linkage and nucleotides downstream of this and the second site is within the loop region. The addition of manganese ions caused recovery of the rate of reaction for phosphorothioate substitutions between A621 and A622 and U631 and C632, suggesting that these two phosphate groups may serve as ligands for two metal ions. In contrast, significant manganese rescue was not observed for the scissile phosphate diester linkage implying that electrophilic catalysis by metal ions is unlikely to contribute to VS ribozyme catalysis. In addition, an increase in the reaction rate of the unmodified VS ribozyme was observed when a mixture of magnesium and manganese ions acted as the cofactor. One possible explanation for this effect is that the cleavage reaction of the VS ribozyme is rate limited by a metal dependent docking of the substrate on the ribozyme.     

The Varkud satellite ribozyme

The VS ribozyme is the largest nucleolytic ribozyme, for which there is no crystal structure to date. The ribozyme consists of five helical sections, organized by two three-way junctions. The global structure has been determined by solution methods, particularly FRET. The substrate stem-loop binds into a cleft formed between two helices, while making a loop-loop contact with another section of the ribozyme. The scissile phosphate makes a close contact with an internal loop (the A730 loop), the probable active site of the ribozyme. This loop contains a particularly critical nucleotide A756. Most changes to this nucleotide lead to three-orders of magnitude slower cleavage, and the Watson-Crick edge is especially important. NAIM experiments indicate that a protonated base is required at this position for the ligation reaction. A756 is thus a strong candidate for nucleobase participation in the catalytic chemistry.     

Addition of an Extra Substrate Binding Site and Partial Destabilization of Stem Structures in HDV Ribozyme Give Rise to High Sequence-Specificity for its Target RNA

Because the substrate binding site (P1) of HDV ribozyme consists of only seven nucleotides, cleavage of undesired RNA is likely to occur when applied for a specific long RNA target such as mRNA. To overcome this problem, we designed modified trans-acting HDV ribozymes with an extra substrate-binding site (P5) in addition to the original binding site (P1). By inserting an additional seven base-pair stem (P5 stem) into the J1/2 single-stranded region of the ribozyme core system and partial destabilization of the P2 or P4 stem, we succeeded in preparation of new HDV ribozymes that can cleave the target RNA depending on the formation of P5 stem. Moreover, the ribozyme with a six-nucleotide P1 site was able to distinguish the substrate RNA with a complete match from that with a single mismatch in the P1 region. These results suggest that the HDV ribozyme system is useful for the application in vivo.     

Imino proton NMR analysis of HDV ribozymes: nested double pseudoknot structure and Mg2+ ion-binding site close to the catalytic core in solution

Minimized trans-acting HDV ribozyme systems consisting of three (Rz-3) and two (Rz-2) RNA strands were prepared and their folding conformations were analyzed by NMR spectroscopy. The guanosine residues in one of the enzyme components of Rz-3 were labeled with ¹³C and ¹⁵N. Imino proton signals were assigned by analysis of NOESY and HSQC spectra. The results are consistent with the nested double pseudoknot model, which contains novel base pairs (P1.1), as observed in the crystal structure of a genomic HDV ribozyme. The NOE connectivities suggest an additional G:G pair at the bottom of P1.1 and at the top of P4. The effects of temperature and Mg²⁺ ions on base pairs for Rz-3 were examined. The temperature variation experiment on Rz-3 showed that P3 is the most stable and that P1.1 is as stable as P1 and P2. The imino proton signals of the G:U pair at the bottom of P1 and the top of P1.1, which are close to the cleavage site, showed the largest changes upon Mg²⁺ titration of Rz-3. The results suggest that the catalytic Mg²⁺ ion binds to the pocket formed by P1 and L3.     

Structural dynamics of precursor and product of the RNA enzyme from the hepatitis delta virus as revealed by molecular dynamics simulations

The hepatitis delta virus (HDV) ribozyme is a self-cleaving RNA enzyme involved in the replication of a human pathogen, the hepatitis delta virus. Recent crystal structures of the precursor and product of self-cleavage, together with detailed kinetic analyses, have led to hypotheses on the catalytic strategies employed by the HDV ribozyme. We report molecular dynamics (MD) simulations (approximately 120 ns total simulation time) to test the plausibility that specific conformational rearrangements are involved in catalysis. Site-specific self-cleavage requires cytidine in position 75 (C75). A precursor simulation with unprotonated C75 reveals a rather weak dynamic binding of C75 in the catalytic pocket with spontaneous, transient formation of a H-bond between U-1(O2') and C75(N3). This H-bond would be required for C75 to act as the general base. Upon protonation in the precursor, C75H+ has a tendency to move towards its product location and establish a firm H-bonding network within the catalytic pocket. However, a C75H+(N3)-G1(O5') H-bond, which would be expected if C75 acted as a general acid catalyst, is not observed on the present simulation timescale. The adjacent loop L3 is relatively dynamic and may serve as a flexible structural element, possibly gated by the closing U20.G25 base-pair, to facilitate a conformational switch induced by a protonated C75H+. L3 also controls the electrostatic environment of the catalytic core, which in turn may modulate C75 base strength and metal ion binding. We find that a distant RNA tertiary interaction involving a protonated cytidine (C41) becomes unstable when left unprotonated, leading to disruptive conformational rearrangements adjacent to the catalytic core. A Na ion temporarily compensates for the loss of the protonated hydrogen bond, which is strikingly consistent with the experimentally observed synergy between low pH and high Na+ concentrations in mediating residual self-cleavage of the HDV ribozyme in the absence of divalents.     

Single VS ribozyme molecules reveal dynamic and hierarchical folding toward catalysis

Non-coding RNAs of complex tertiary structure are involved in numerous aspects of the replication and processing of genetic information in many organisms; however, an understanding of the complex relationship between their structural dynamics and function is only slowly emerging. The Neurospora Varkud Satellite (VS) ribozyme provides a model system to address this relationship. First, it adopts a tertiary structure assembled from common elements, a kissing loop and two three-way junctions. Second, catalytic activity of the ribozyme is essential for replication of VS RNA in vivo and can be readily assayed in vitro. Here we exploit single molecule FRET to show that the VS ribozyme exhibits previously unobserved dynamic and heterogeneous hierarchical folding into an active structure. Readily reversible kissing loop formation combined with slow cleavage of the upstream substrate helix suggests a model whereby the structural dynamics of the VS ribozyme favor cleavage of the substrate downstream of the ribozyme core instead. This preference is expected to facilitate processing of the multimeric RNA replication intermediate into circular VS RNA, which is the predominant form observed in vivo.     

Catalytic cleavage of cis- and trans-acting antigenomic delta ribozymes in the presence of various divalent metal ions

Catalytic activity of four structural variants of the antigenomic delta ribozyme, two cis- and two trans-acting, has been compared in the presence of selected divalent metal ions that effectively support catalysis. The ribozymes differ in regions that are not directly involved in formation of the ribozyme active site: the region immediately preceding the catalytic cleavage site, the P4 stem and a stretch of the viral RNA sequence extending the minimal ribozyme sequence at its 3′-terminus. The variants show high cleavage activity in the presence of Mg²⁺, Ca²⁺ and Mn²⁺, lower with Co²⁺ and Sr²⁺ and some variants are also active with Cd²⁺ and Zn²⁺ ions. In the presence of a particular metal ion the ribozymes cleave, however with different initial rates, according to pseudo-first or higher order kinetics and to different final cleavage extents. On the other hand, relatively small differences are observed in the reactions induced by various metal ions. The cleavage of trans-acting ribozymes induced by Mg²⁺ is partially inhibited in the presence of Na⁺, spermidine and some other divalent metal ions. The inert Co(NH₃)₆³⁺ complex is unable to support catalysis, as reported earlier for the genomic ribozyme. The results are discussed in terms of the influence of structural elements peripheral to the ribozyme active site on its cleavage rate and efficiency as well as the role of metal ions in the cleavage mechanism. Some implications concerning further studies and possible applications of delta ribozymes are also considered.