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.     

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.     

General acid catalysis by the hepatitis delta virus ribozyme

Recent crystallographic and functional analyses of RNA enzymes have raised the possibility that the purine and pyrimidine nucleobases may function as general acid-base catalysts. However, this mode of nucleobase-mediated catalysis has been difficult to establish unambiguously. Here, we used a hyperactivated RNA substrate bearing a 5'-phosphorothiolate to investigate the role of a critical cytosine residue in the hepatitis delta virus ribozyme. The hyperactivated substrate specifically suppressed the deleterious effects of cytosine mutations and pH changes, thereby linking the protonation of the nucleobase to leaving-group stabilization. We conclude that the active-site cytosine provides general acid catalysis, mediating proton transfer to the leaving group through a protonated N3-imino nitrogen. These results establish a specific role for a nucleobase in a ribozyme reaction and support the proposal that RNA nucleobases may function in a manner analogous to that of catalytic histidine residues in protein enzymes.     

A circular trans-acting hepatitis delta virus ribozyme.

A circular trans-acting ribozyme designed to adopt the motif of the hepatitis delta virus (HDV) trans-acting ribozyme was produced. The circular form was generated in vitro by splicing a modified group I intron precursor RNA in which the relative order of the 5' and 3' splice sites, flanking the single HDV-like ribozyme sequence-containing exon, is reversed. Trans-cleavage activity of the circular HDV-like ribozyme was comparable to linear permutations of HDV ribozymes containing the same core sequence, and was shown not to be due to linear contaminants in the circular ribozyme preparation. In nuclear and cytoplasmic extracts from HeLa cells, the circular ribozyme had enhanced resistance to nuclease degradation relative to a linear form of the ribozyme, suggesting that circularization may be a viable alternative to chemical modification as a means of stabilizing ribozymes against nuclease degradation.     

Nucleotides -1 to -4 of hepatitis delta ribozyme substrate increase the specificity of ribozyme cleavage

In the past, the use of delta ribozyme as a therapeutic tool was limited because substrate specificity was thought to be determined by only 8 nucleotides. Recently, we have accumulated evidence suggesting that the substrate sequence upstream of the cleavage site, which is not involved in the binding with the delta ribozyme, appears to be essential in the selection of an appropriate cleavage site. To understand the role of this region in efficient cleavage, we synthesized a collection of small substrates that possessed single and multiple mutations in positions -1 to -4 and determined the kinetic parameters of their cleavage using a model antigenomic delta ribozyme. Some substrates were found to be uncleavage, whereas others showed >60-fold difference in relative specificity between the least and most efficiently cleaved substrates. The base at each position from -1 to -4 contributes differently to the ability of a substrate to be cleaved. An optimal sequence for positions -1 to -4 was determined to be -1HRHY(-4) (H = U, C, or A). These results shed light on new features that contribute to the substrate requirement of delta ribozyme cleavage and should increase interest in the use of this unique ribozyme.     

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.     

A novel structural rearrangement of hepatitis delta virus antigenomic ribozyme

A bioinformatic covariation analysis of a collection of 119 novel variants of the antigenomic, self-cleaving hepatitis delta virus (HDV) RNA motif supported the formation of all of the Watson–Crick base pairs (bp) of the catalytic centre except the C19–G81 pair located at the bottom of the P2 stem. In fact, a novel Watson–Crick bp between C19 and G80 is suggested by the data. Both chemical and enzymatic probing demonstrated that initially the C19–G81 pair is formed in the ribozyme (Rz), but upon substrate (S) binding and the formation of the P1.1 pseudoknot C19 switches its base-pairing partner from G81 to G80. As a result of this finding, the secondary structure of this ribozyme has been redrawn. The formation of the C19–G80 bp results in a J4/2 junction composed of four nucleotides, similar to that seen in the genomic counterpart, thereby increasing the similarities between these two catalytic RNAs. Additional mutagenesis, cleavage activity and probing experiments yield an original characterization of the structural features involving the residues of the J4/2 junction.     

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.     

Properties of antigenomic hepatitis delta virus ribozyme cis- and trans- analogs

A series of permuted variants of antigenomic HDV ribozyme and trans-acting variants were constructed. The catalytic activity study of the ribozymes has shown that all the variants were capable of self-cleaving with equally biphasic kinetics. Ribonuclease and Fe(II)-EDTA cleavage have provided evidence that all designed ribozymes fold according to the pseudoknot model and the conformations of the initial and cleaved ribozyme are different. A scheme of HDV ribozyme self-cleavage reaction was suggested. The role of hydrogen bonds in the reaction was evaluated by substitution of ribose in the ribozyme for deoxyribose. It was found that the 2'-OH group of U23 and C27 is critical for the reaction to occur; the 2'-OH group of U32 and U39 is important, while 2'-OH groups of other nucleotides of loop 3, stem 4 and stem 1 are unimportant for the cleavage activity.     

Direct selection for ribozyme cleavage activity in cells

Selection may prove to be a powerful tool for the generation of functional RNAs for in vivo genetic regulation. However, traditional in vitro selection schemes do not mimic physiological conditions, and in vivo selection schemes frequently use small pool sizes. Here we describe a hybrid in vitro/in vivo selection scheme that overcomes both of these disadvantages. In this new method, PCR-amplified expression templates are transfected into mammalian cells, transcribed hammerhead RNAs self-cleave, and the extracted, functional hammerhead ribozyme species are specifically amplified for the next round of selection. Using this method we have selected a number of cis-cleaving hammerhead ribozyme variants that are functional in vivo and lead to the inhibition of gene expression. More importantly, these results have led us to develop a quantitative, kinetic model that can be used to assess the stringency of the hybrid selection scheme and to direct future experiments.     

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.     

Intracellular cleavage and ligation of hepatitis delta virus genomic RNA: regulation of ribozyme activity by cis-acting sequences and host factors.

During replication, a ribozyme within the genomic RNA of hepatitis delta virus cleaves multimeric precursors to release a unit-length linear intermediate. Intramolecular ligation of this intermediate produces the circular genomic RNA. Although one copy of the ribozyme is reconstituted by such ligation, it does not subsequently cleave and destroy the circular conformation. We have identified cis-acting attenuator sequences that prevent self-cleavage of the circular product by base pairing with and inactivating the ribozyme. Furthermore, we have shown that during the initial processing of the multimeric precursor RNA, host-specific factors activate the ribozyme by preventing its association with the attenuator sequences. Thus, we demonstrate a novel switching mechanism that regulates ribozyme activity inside the cell.     

Requirement for canonical base pairing in the short pseudoknot structure of genomic hepatitis delta virus ribozyme

The tertiary structure of the 3′-cleaved product of the genomic hepatitis delta virus (HDV) ribozyme was solved by X-ray crystallographic analysis. In this structure, three single-stranded regions (SSrA, -B and -C) interact intricately with one another via hydrogen bonds between nucleotide bases, phosphate oxygens and 2′-OHs to form a nested double pseudoknot structure. Among these interactions, two Watson–Crick (W–C) base pairs, 726G–710C and 727G–709C, that form between SSrA and SSrC (P1.1) seem to be especially important for compact folding. To characterize the importance of these base pairs, ribozymes were subjected to in vitro selection from a pool of RNA molecules randomly substituted at positions 709, 710, 726 and 727. The results establish the importance of the two W–C base pairs for activity, although some mutants are active with one G–C base pair. In addition, the kinetic parameters were analyzed in all 16 combinations with two canonical base pairs. Comparison of variant ribozymes with the wild-type ribozyme reveals that the difference in reaction rates for these variants (ΔΔG^‡) is not simply accounted for by the differences in the stability of P1.1 (ΔΔG⁰₃₇). The role played by Mg²⁺ ions in formation of the P1.1 structure is also discussed.     

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.     

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.     

Mapping of accessible sites for oligonucleotide hybridization on hepatitis delta virus ribozymes

Semi-random libraries of DNA 6mers and RNase H digestion were applied to search for sites accessible to hybridization on the genomic and antigenomic HDV ribozymes and their 3′ truncated derivatives. An approach was proposed to correlate the cleavage sites and most likely sequences of oligomers, members of the oligonucleotide libraries, which were engaged in the formation of RNA–DNA hybrids. The predicted positions of oligomers hybridizing to the genomic ribozyme were compared with the fold of polynucleotide chain in the ribozyme crystal structure. The data exemplified the crucial role of target RNA structural features in the binding of antisense oligonucleotides. It turned out that cleavages were induced if the bound oligomer could adapt an ordered helical conformation even when it required partial penetration of an adjacent double-stranded region. The major features of RNA structure disfavoring hybridization and/or RNase H hydrolysis were sharp turns of the polynucleotide chain and breaks in stacking interactions of bases. Based on the predicted positions of oligomers hybridizing to the antigenomic ribozyme we chose and synthesized four antisense DNA 6mers which were shown to direct hydrolysis in the desired, earlier predicted regions of the molecule.     

Long-range impact of peripheral joining elements on structure and function of the hepatitis delta virus ribozyme

The HDV ribozyme is an RNA enzyme from the human pathogenic hepatitis delta virus (HDV) that has recently also been identified in the human genome. It folds into a compact, nested double-pseudoknot. We examined here the functional relevance of the capping loop L4 and the helical crossover J1/2, which tightly interlace the two helical stacks of the ribozyme. Peripheral structural elements such as these are present in cis-acting, but not trans-acting ribozymes, which may explain the order-of-magnitude decrease in cleavage activity observed in trans-acting ribozymes with promise in gene therapy applications. Comparison of a systematic set of cis- and trans-acting HDV ribozymes shows that the absence of either L4 or J1/2 significantly and independently impacts catalytic activity. Using terbium(III) footprinting and affinity studies, as well as distance measurements based on time-resolved fluorescence resonance energy transfer, we find that J1/2 is most important for conferring structural properties similar to those of the cis-acting ribozyme. Our results are consistent with a model in which removal of either a helical crossover or surprisingly a capping loop induces greater dynamics and expansion of the catalytic core at long range, impacting local and global folding, as well as catalytic function.     

Assessment of disparate structural features in three models of the hepatitis delta virus ribozyme.

Three models for the secondary structure of the hepatitis delta virus (HDV) antigenomic self-cleaving RNA element were tested by site-directed mutagenesis. Two models in which bases 5' to the cleavage site are paired with sequence at the 3' end of the element were both inconsistent with the data from the mutagenesis. Specifically, mutations in the 3' sequence which decrease self-cleavage activity could not be compensated by base changes in the 5' sequence as predicted by these models. The evidence was consistent with a third model in which the 3' end pairs with a portion of a loop within the ribozyme sequence to generate a pseudoknot structure. This same pairing was also required to generate higher rates of cleavage in trans with a 15-mer ribozyme, thus ruling out a proposed hammerhead-like 'axehead' model for the HDV ribozyme.