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.     

Presence of a coordinated metal ion in a trans-acting antigenomic delta ribozyme.

We have investigated the cleavage induced by metal ions in an antigenomic form of a trans-acting delta ribozyme. A specific Mg(2+)-induced cleavage at position G(52)at the bottom of the P2 stem was observed to occur solely within catalytically active ribozyme-substrate complexes (i.e. those that performed the essential conformational transition step). Only the divalent cations which support catalytic activity permitted the detection of specific induced cleavages in this region. Using various mutant ribozymes and substrates, we demonstrated a correlation between enzymatic activity and the Mg(2+)-induced cleavage pattern. We show that the efficiency of the coordination of the magnesium to its binding site is related to the nature of the base pair in the middle of the P1 stem (i.e. Rz(23)-S(8)). Together with additional evidence from nuclease probing experiments that indicates the occurrence of a structural rearrangement involving the bottom of the P2 stem upon formation of the P1 helix, these results show that an intimate relationship exists between the folding and the catalytic activity of the delta ribozyme.     

Inhibition of the catalytic activity of trans-acting antigenomic delta ribozyme by selected antibiotics and their Cu2+ complexes

The effect of selected 10 antibiotics and their complexes with Cu(2+) ions on the catalytic activity of the trans-acting antigenomic delta ribozyme was investigated. Sisomicin, vancomycin, and actinomycin D displayed weak inhibitory properties. However, much stronger effects were detected with complexes of these antibiotics with Cu(2+) ions. The strongest inhibition was observed with actinomycin D-Cu(2+) complex, for which the calculated K(i) value was reduced ca. 35-fold upon metal ion complexation. We postulate that the antibiotic-Cu(2+) complexes are guided to the ribozyme metal ion binding site(s) presumably displacing the catalytically important metal ion(s). Moreover, we assume that, once positioned in appropriate distances to RNA phosphate groups and bases, the coordinated Cu(2+) ions become positively charged factors that enhance the affinity of the antibiotics to the ribozyme. These observations indicate that coordination of metal ions to antibiotics substantially changes their properties which might also have a biological relevance inside the cell.     

Analysis of the cleavage reaction of a trans-acting human hepatitis delta virus ribozyme.

The cleavage reaction catalyzed by the trans -acting genomic ribozyme of human hepatitis delta virus (HDV) was analyzed with a 13mer substrate (R13) and thio-substituted [SR13(Rp) and SR13(Sp)] substrates under single-turnover conditions. The cleavage of RNA by the trans -acting HDV ribozyme proceeded as a first order reaction. The logarithm of the rate of cleavage (kclv) increased linearly (with a slope of approximately 1) between pH 4.0 and 6.0, an indication that a single deprotonation reaction occurred. This result suggests that kclv reflects the rate of the chemical cleavage step, at least around pH 5. The amount of active complex with the SR13(Sp) substrate was almost as large as with R13 (60-80%), whereas the amount of the corresponding active complex formed with the SR13(Rp) substrate was, at most, 20% of this value (with 0.5-100 mM Mg2+ions) at pH 5.0. Nonetheless, the value of kclv for all substrates was almost the same (0.4-0.5 min-1). Neither a 'thio effect' nor a 'Mn2+rescue effect' were observed. These results suggest that Mg2+ions do not interact with pro-R oxygen directly but are essential to the formation of the active complex of the ribozyme and its substrate.     

Modification interference approach to detect ribose moieties important for the optimal activity of a ribozyme.

A new approach for modification interference studies is presented. It involves the use of phosphorothioates as a handle to analyze any desired base or sugar modification. This method was applied to identify ribose and phosphate moieties which could be important in the pre-tRNA recognition of E. coli RNase P RNA (M1 RNA). The utility of this technique was confirmed by detecting the inhibitory effect of a deoxyribose in the 5'-flank (position-1). This site was already known to interfere with RNase P cleavage, if modified. We have analyzed pre-tRNA(Tyr) and pre-tRNA(Phe) and found different interference patterns for both tRNAs. Two unpaired regions were involved in both pre-tRNAs. Phosphorothioates interfered at the transition between acceptor- and D-arms. The results with deoxythymidines in the T-loop indicated that deoxyribose moieties or the extra methyl group in thymidine could interfere with RNAse P cleavage. These data suggest that even in complete pre-tRNAs, only a few intact ribonucleotides are important in the substrate recognition by RNase P. We have demonstrated the potential of this new approach which offers many future applications in all fields involving nucleic acids, for example RNA processing, action of ribozymes, tRNA charging and studies related to DNA promoter recognition.     

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.     

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.     

Mutational analysis of the antigenomic trans-acting delta ribozyme: the alterations of the middle nucleotides located on the P1 stem

Our previous report on delta ribozyme cleavage using a trans -acting antigenomic delta ribozyme and a collection of short substrates showed that the middle nucleotides of the P1 stem, the substrate binding site, are essential for the cleavage activity. Here we have further investigated the effect of alterations in the P1 stem on the kinetic and thermodynamic parameters of delta ribozyme cleavage using various ribozyme variants carrying single base mutations at putative positions reported. The kinetic and thermodynamic values obtained in mutational studies of the two middle nucleotides of the P1 stem suggest that the binding and active sites of the delta ribozyme are uniquely formed. Firstly, the substrate and the ribozyme are engaged in the formation of a helix, known as the P1 stem, which may contain a weak hydrogen bond(s) or a bulge. Secondly, a tertiary interaction involving the base moieties in the middle of the P1 stem likely plays a role in defining the chemical environment. As a con-sequence, the active site might form simultaneously or subsequently to the binding site during later steps of the pathway.     

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.     

Mutagenesis analysis of a hepatitis delta virus genomic ribozyme.

We conducted extensive mutagenesis analysis on a hepatitis delta virus (HDV) genomic ribozyme to study the sequence specificity of certain region and to derive the secondary structure associated with the catalytic core. The results confirmed that the autocatalytic domain of HDV genomic RNA contained four base-pairing regions as predicted in the 'pseudo-knot' model [Perrotta & Been (1990) Nature 350, 434-436]. The size and sequence of one of the base-pairing regions, i. e. stem-and-loop, could be flexible. Helix 3 and the first basepair of helix 1 required specific sequence to retain self-cleavage activity. The structural requirement of helix 2 was less stringent than the other base-pairing regions. Moreover, the size of helix 1 affected self-cleavage whereas the length of hinge could be variable even though the first three residues of hinge had stringent sequence requirement.     

Cations and hydration in catalytic RNA: molecular dynamics of the hepatitis delta virus ribozyme

The hepatitis delta virus (HDV) ribozyme is an RNA enzyme from the human pathogenic HDV. Cations play a crucial role in self-cleavage of the HDV ribozyme, by promoting both folding and chemistry. Experimental studies have revealed limited but intriguing details on the location and structural and catalytic functions of metal ions. Here, we analyze a total of approximately 200 ns of explicit-solvent molecular dynamics simulations to provide a complementary atomistic view of the binding of monovalent and divalent cations as well as water molecules to reaction precursor and product forms of the HDV ribozyme. Our simulations find that an Mg2+ cation binds stably, by both inner- and outer-sphere contacts, to the electronegative catalytic pocket of the reaction precursor, in a position to potentially support chemistry. In contrast, protonation of the catalytically involved C75 in the precursor or artificial placement of this Mg2+ into the product structure result in its swift expulsion from the active site. These findings are consistent with a concerted reaction mechanism in which C75 and hydrated Mg2+ act as general base and acid, respectively. Monovalent cations bind to the active site and elsewhere assisted by structurally bridging long-residency water molecules, but are generally delocalized.     

Kinetic scheme for intermolecular RNA cleavage by a ribozyme derived from hepatitis delta virus RNA

A minimal kinetic mechanism for a trans-acting ribozyme derived from the HDV antigenomic RNA self-cleaving element was established from steady-state, pre-steady-state, single-turnover, and binding kinetics. Rate constants for individual steps, including substrate binding and dissociation, cleavage, and product release and binding, were measured at 37 degrees C at pH 8.0 in 10 mM Mg(2+) using oligonucleotides as either substrates, noncleavable analogues or 3' product mimics. A substrate containing a normal 3',5'-linkage was cleaved with a first-order rate constant (k(2)) of 0.91 min(-)(1). The association rate constant for the substrate to the ribozyme (2.1 x 10(7) M(-)(1) min(-)(1)) was at the lower range of the expected value for RNA duplex formation, and the substrate dissociated with a rate constant (1.4 min(-)(1)) slightly faster than that for cleavage. Thus the binary complex was not at equilibrium with free enzyme and substrate prior to the cleavage step. Following cleavage, product release was kinetically ordered in that the 5' product was released rapidly (>12 min(-)(1)) relative to the 3' product (6.0 x 10(-)(3) min(-)(1)). Rapid 5' product release and lack of a demonstrable binding site for the 5' product could contribute to the difficulty in establishing the ribozyme-catalyzed reverse reaction (ligation). Slow release of the 3' product was consistent with the extremely low turnover under steady-state conditions as 3' product dissociation was rate-limiting. The equilibrium dissociation constant for the substrate was 24-fold higher than that of the 3' cleavage product. A substrate with a 2',5'-linkage at the cleavage site was cleaved with a rate constant (k(2)) of 1.1 x 10(-)(2) min(-)(1). Thus, whereas cleavage of a 3',5'-linkage followed a Briggs-Haldane mechanism, 2', 5' cleavage followed a Michaelis-Menten mechanism.     

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.     

Functional characterization of the SOFA delta ribozyme

Molecular engineering has led to the development of a novel target-dependent riboswitch that increases deltaribozyme fidelity. This delta ribozyme possesses a specific on/off adapter (SOFA) that switches the cleavage activity from off (a "safety lock") to on solely in the presence of the desired RNA substrate. In this report, we investigate the influence of both the structure and the sequence of each domain of the SOFA module. Analysis of the cleavage activity, using a large collection of substrates and SOFA-ribozyme mutants, together with RNase H probing provided several insights into the nature of the sequence and the optimal design of each domain of the SOFA module. For example, we determined that (1) the optimal size of the blocker sequence, which keeps the ribozyme off in the absence of the substrate, is 4 nucleotides (nt); (2) a single nucleotide difference between the substrate and the biosensor domain, which is responsible for the initial binding of the substrate that subsequently switches the SOFA-ribozyme on, is sufficient to cause non-recognition of the appropriate substrate; (3) the stabilizer, which joins the 5' and 3' ends of the SOFA-ribozyme, plays only a structural role; and (4) the optimal spacer sequence, which serves to separate the binding regions of the biosensor and catalytic domain of the ribozyme on the substrate, is from 1 to 5 nt long. Together, these data should facilitate the design of more efficient SOFA-ribozymes with significant potential for many applications in gene-inactivation systems.     

Antigenomic delta ribozyme variants with mutations in the catalytic core obtained by the in vitro selection method

We have used the in vitro selection method to search for catalytically active variants of the antigenomic delta ribozyme with mutations in the regions that constitute the ribozyme active site: L3, J1/4 and J4/2. In the initial combinatorial library 16 nt positions were randomized and the library contained a full representation of all possible sequences. Following ten cycles of selection-amplification several catalytically active ribozyme variants were identified. It turned out that one-third of the variants contained only single mutation G80U and their activity was similar to that of the wild-type ribozyme. Unexpectedly, in the next one-third of the variants the C76 residue, which was proposed to play a crucial role in the ribozyme cleavage mechanism, was mutated. In these variants, however, a cytosine residue was present in a neighboring position to the polynucleotide chain. It shows that the ribozyme catalytic core possesses substantial ‘structural plasticity’ and the capacity of functional adaptation. Four selected ribozyme variants were subjected to more detailed analysis. It turned out that the variants differed in their relative preferences towards Mg²⁺, Ca²⁺ and Mn²⁺ ions. Thus, the functional properties of the variants were dependent on both the structure of their catalytic sites and divalent metal ions performing catalysis.     

Impact of an extruded nucleotide on cleavage activity and dynamic catalytic core conformation of the hepatitis delta virus ribozyme

The self-cleaving hepatitis delta virus (HDV) ribozyme is essential for the replication of HDV, a liver disease causing pathogen in humans. The catalytically critical nucleotide C75 of the ribozyme is buttressed by a trefoil turn pivoting around an extruded G76. In all available crystal structures, the conformation of G76 is restricted by stacking with G76 of a neighboring molecule. To test whether this crystal contact introduces a structural perturbation into the catalytic core, we have analyzed approximately 200 ns of molecular dynamics (MD) simulations. In the absence of crystal packing, the simulated G76 fluctuates between several conformations, including one wherein G76 establishes a perpendicular base quadruplet in the major groove of the adjacent P1 stem. Second-site mutagenesis experiments suggest that the identity of the nucleotide in position 76 (N76) indeed contributes to the catalytic activity of a trans-acting HDV ribozyme through its capacity for hydrogen bonding with P1. By contrast, in the cis-cleaving genomic ribozyme the functional relevance of N76 is less pronounced and not correlated with the P1 sequence. Terbium(III) footprinting and additional MD show that the activity differences between N76 mutants of this ribozyme are related instead to changes in average conformation and modified cross-correlations in the trefoil turn.     

Detailed analysis of base preferences at the cleavage site of a trans-acting HDV ribozyme: a mutation that changes cleavage site specificity.

In our previous attempt at in vitro selection of a trans - acting human hepatitis delta virus (HDV) ribozyme, we found that one of the variants, G10-68-725G, cleaved a 13 nt substrate, HDVS1, at two sites [Nishikawa,F., Kawakami,J., Chiba,A., Shirai,M., Kumar,P.K.R. and Nishikawa,S. (1996) Eur. J. Biochem., 237, 712-718]. One site was the normal cleavage site and the other site was shifted 1 nt toward the 3'-end. To clarify the interactions between nucleotides around the cleavage site of the trans -acting HDV ribozyme, we analyzed the efficiency of the reaction for every possible base pair between the substrate and the ribozyme at positions -1 (-1N:726N) and +1 (+1N:725N) relative to the cleavage site using the genomic HDV ribozyme, TdS4(Xho), and derivatives of the most active variant, G10-68. These mutagenesis analyses revealed that the +1 base of the substrate affects the structure of the catalytic core in the complex with G10-68-725G, substrate and divalent metal ions, and it shifts the cleavage site. In a comparison with other variants of the trans -acting HDV ribozyme, we found that this cleavage site shift occurred only with G10-68-725G.