Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0019158 (hepatitis)
 30,205 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Self-cleaving sequences or ribozymes from the hepatitis delta virus (HDV) genomic RNA and its complement form similar secondary structures that suggest a core region and potential active site composed of "single-stranded" sequences. However, there is little data on tertiary interactions in these ribozymes, therefore structural features were investigated using cross-linking and hydroxyl radical cleavage. Cross-links in cis and trans forms of the antigenomic RNA were generated using the photoactivatable azidophenacyl group tethered to the cleavage site phosphate. Specific cross-links formed to J4/2, and to the 3' sides of P3 and L3. Different sites were cross-linked in low salt or monovalent cations versus divalent cations, suggesting a metal ion-dependent conformational change near the cleavage site. The solvent-inaccessible regions of both the genomic and antigenomic ribozymes were revealed by cleavage in Fe(II)-EDTA. In Mg2+, backbone segments most strongly protected from solvent-based hydroxyl radicals were mapped to J4/2 and parts of L3. Similar patterns of protection were seen in trans-acting ribozymes bound to a product oligonucleotide. These data provide evidence for a common tertiary structure for the HDV ribozymes. They would be consistent with a model in which the end of P1, including the cleavage site phosphate and the nucleotide 5' to the cleavage site, is positioned in an active site pocket or cleft formed by the three single-stranded regions, L3, J4/2, and J1/4.
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PMID:Hepatitis delta virus ribozymes fold to generate a solvent-inaccessible core with essential nucleotides near the cleavage site phosphate. 878 96

Properties of a hepatitis delta virus (HDV) RNA ribozyme system, which consists of three RNA oligomer strands (substrate 8-mer; enzyme 16-mer plus 35-mer) and contains a hybrid sequence of genomic and antigenomic RNA cores, are reported. Effects of Mg2+ concentration, divalent metal ion species, pH, and temperature on the cleavage activity were examined. The substrate cleavage activity increased with increasing Mg2+ concentration (0-100 mM). Ca2+ and Mn2+ ions were the most effective divalent cations and Mg2+ was less effective. The cleavage activity increased with increasing pH (5-7.5). The optimum temperature for the cleavage activity was 25-40 degrees C. The Mg2+ concentration, pH and temperature dependencies are different from those reported for the single-strand ribozymes (about 90-mer) although the divalent metal ion preference is very similar. Conformational change induced by Mg2+ ion titration was monitored by CD. The CD data and the activity-Mg2+ concentration data were analyzed by curve-fitting analysis using equations derived for multiple metal ion binding mechanisms. The data can be explained by a model in which three Mg2+ ions bind to one ribozyme unit.
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PMID:Properties of hepatitis delta virus ribozyme, which consists of three RNA oligomer strands. 935 86

The antigenomic RNA of hepatitis delta virus (HDV) can form a short duplex, P2a, in which a four-nucleotide sequence within the self-cleaving domain pairs with a sequence just outside the previously defined 3'-boundary of the ribozyme. Both sequences that would participate in forming P2a were previously determined to be non-essential for self-cleavage activity. Ribozymes able to form P2a were less active than those lacking the 3' P2a sequence when preincubated under the standard low-Na+ conditions. Chemical probing of the RNA correlated base-pairing in P2a with this inhibition. Furthermore, mutagenesis and 3' truncation experiments mapped the inhibitory sequence to P2a. However, raising the NaCl concentration in the preincubation prior to adding Mg2+ reversed the inhibitory effect. Moreover, with NaCl preincubation, the P2a-containing ribozyme was more active than an otherwise identical ribozyme lacking the 3' P2a sequence. Non-denaturing gels provided evidence for alternative conformations of the P2a-containing precursor with only the faster-migrating species correlating with the active form. A difference in the temperature-dependence for the rate of cleavage of the P2a-containing ribozyme with and without NaCl, together with a difference in the melting behavior of the RNA in NaCl with and without P2a, suggested that P2a favors the native structure in NaCl. Many derivatives of the HDV ribozymes form inactive conformers; however, this study reveals details of a specific structure that stabilizes both inactive and active conformations of the HDV ribozyme.
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PMID:A toggle duplex in hepatitis delta virus self-cleaving RNA that stabilizes an inactive and a salt-dependent pro-active ribozyme conformation. 964 43

The natural substrate cleaved by the hepatitis delta virus (HDV) ribozyme contains a 3',5'-phosphodiester linkage at the cleavage site; however, a 2',5'-linked ribose-phosphate backbone can also be cleaved by both trans-acting and self-cleaving forms of the HDV ribozyme. With substrates containing either linkage, the HDV ribozyme generated 2',3'-cyclic phosphate and 5'-hydroxyl groups suggesting that the mechanisms of cleavage in both cases were by a nucleophilic attack on the phosphorus center by the adjacent hydroxyl group. Divalent metal ion was required for cleavage of either linkage. However, although the 3',5'-linkage was cleaved slightly faster in Ca2+ than in Mg2+, the 2',5'-linkage was cleaved in Mg2+ (or Mn2+) but not Ca2+. This dramatic difference in metal-ion specificity is strongly suggestive of a crucial metal-ion interaction at the active site. In contrast to the HDV ribozymes, cleavage at a 2',5'-phosphodiester bond was not efficiently catalyzed by the hammerhead ribozyme. The relaxed linkage specificity of the HDV ribozymes may be due in part to lack of a rigid binding site for sequences 5' to the cleavage site.
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PMID:Ribozyme cleavage of a 2,5-phosphodiester linkage: mechanism and a restricted divalent metal-ion requirement. 1049 15

Human hepatitis delta virus (HDV) ribozyme can catalyze self-cleavage reaction in the presence of Mg2+ ions, yielding products with 2',3'-cyclic phosphate and 5'-OH termini as do hammerhead and hairpin ribozymes. Recently, the tertiary structure of 3'-cleaved product of genomic HDV ribozyme was solved by X-ray crystallographic analysis. In this structure three single-stranded regions (SSrA, -B and -C) interacts intricately with hydrogen bonds between bases, phosphate oxygens and 2'-OHs to form nested double pseudoknot structure. Especially two Watson-Crick base pairs, 726G-710C and 727G-709C, between SSrA and SSrC, seems to be important for compact folding. To characterize the necessity of the two base pairs, we performed in vitro selection of active ribozymes using random RNA pool which mutated at 709, 710, 726 and 727. The result indicates that basically one G-C base pair is necessary for the activity.
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PMID:Importance of short pseudoknot base pairs between two single-stranded regions of HDV ribozyme. 1078 Apr 92

Self-cleavage of the genomic and antigenomic ribozymes from hepatitis delta virus (HDV) requires divalent cation for optimal activity. Recently, the HDV genomic ribozyme has been shown to be active in NaCl in the absence of added divalent metal ion at low pH (apparent pKa 5.7). However, we find that the antigenomic ribozyme is 100 to 1000-fold less active under similar conditions. With deletion of a three-nucleotide sequence (C41-A42-A43) unique to the genomic ribozyme, the rate constant for cleavage decreased substantially, while activity of the antigenomic ribozyme was enhanced by introducing a CAA sequence. From the crystal structure, it has been proposed that C41 in this sequence is protonated. To investigate a possible connection between activity at low pH and protonation of C41, mutations were made that were predicted to either eliminate protonation or alter the nature of the tertiary interaction upon protonation. In the absence of added Mg2+, these mutations reduced activity and eliminated the observed pH-rate dependence. Thermal denaturation studies revealed a pH-sensitive structural feature in the genomic ribozyme, while unfolding of the mutant ribozymes was pH-independent. We propose that, in the absence of added Mg2+, protonation of C41 contributes to enhanced activity of the HDV genomic ribozyme at low pH.
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PMID:A pH-sensitive RNA tertiary interaction affects self-cleavage activity of the HDV ribozymes in the absence of added divalent metal ion. 1116 13

Human hepatitis delta (HDV) ribozyme is one of small ribozymes, such as hammerhead and hairpin ribozymes, etc. Its secondary structure shows pseudoknot structure composed of four stems (I to IV) and three single-stranded regions (SSrA, -B and -C). The 3D structure of 3'-cleaved product of genomic HDV ribozyme provided extensive information about tertiary hydrogen bonding interactions between nucleotide bases, phosphate oxygens and 2'OHs including new stem structure P1.1. To analyze the role of these hydrogen bond networks in the catalytic reaction, site-specific atomic-level modifications (such as deoxynucleotides, deoxyribosyl-2-aminopurine, deoxyribosylpurine, 7-deaza-ribonucleotide and inosine) were incorporated in the smallest trans-acting HDV ribozyme (47-mer). Kinetic analysis of these ribozyme variants demonstrated the importance of the two W-C base pairs of P1.1 for cleavage; in addition, the results suggest that all hydrogen bond interactions detected in the crystal structure involving 2'-OH and N7 atoms are present in the active ribozyme structure. In most of the variants, the relative reduction in kobs caused by substitution of the 2'-OH group correlated with the number of hydrogen bonds affected by the substitution. However G74 and C75 may have more than one hydrogen bond involving the 2'-OH in both the trans- and cis-acting HDV ribozyme. Moreover, in variants in which N7 was deleted, kobs was reduced 5- to 15-fold, it may suggest that N7 assists in coordinating Mg2+ ions or water molecules which bind with weak affinity in the active structure.
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PMID:Site-specific modification of functional groups in genomic hepatitis delta virus (HDV) ribozyme. 1244 67

A three-strand ribozyme, a derivative of antigenomic hepatitis delta virus (HDV) ribozyme, which consists of subfragments of 16 (L), 17 (S), and 33 nucleotides (B), has been constructed. The ternary B-L-S complex formed by the subfragments in stoichiometric ratio was able to catalyze a self-cleavage reaction. Kinetics of this reaction exhibited biphasic behavior and the same parameters as in the case of natural cis-ribozyme. Study of kinetics of reaction initiated by adding various reaction components and the study of binary complex formation between subfragments B and L, B and S, and also ternary B-L-S complex formation revealed that: 1) in the presence of Mg2+, B and S form a stoichiometric complex, L and S do not form complex at all, while B and L form 2 types of complexes, probably B-L and 2B-L; and addition of S subfragment prevented the formation of the latter complex; 2) the reaction initiated by S subfragment proceeds much slower than that initiated by other components pointing to the possibility that in the absence of S L may form a nonproductive complex with B, which is slowly displaced by S followed by productive ternary complex formation. Dissociation constants for binary B-L, B-S and ternary B-L-S complexes have been estimated.
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PMID:Properties of antigenomic hepatitis delta virus ribozyme that consists of three RNA oligomer strands. 1464 Sep 66

The hepatitis delta virus (HDV) ribozyme is a self-cleaving RNA that resides in the HDV genome and regulates its replication. The native fold of the ribozyme is complex, having two pseudoknots. Earlier work implicated four non-native pairings in slowing pseudoknot formation: Alt 1, Alt 2, Alt 3, and Alt P1. The goal of the present work was design of a kinetically simplified and maximally reactive construct for in vitro mechanistic and structural studies. The initial approach chosen was site-directed mutagenesis in which known alternative pairings were destabilized while leaving the catalytic core intact. Based on prior studies, the G11C/U27Delta double mutant was prepared. However, biphasic kinetics and antisense oligonucleotide response trends opposite those of the well-studied G11C mutant were observed suggesting that new alternative pairings with multiple registers, termed Alt X and Alt Y, had been created. Enzymatic structure mapping of oligonucleotide models supported this notion. This led to a model wherein Alt 2 and the phylogenetically conserved Alt 3 act as "folding guides", facilitating folding of the major population of the RNA molecules by hindering formation of the Alt X and Alt Y registers. Attempts to eliminate the strongest of the Alt X pairings by rational design of a quadruple mutant only resulted in more complex kinetic behavior. In an effort to simultaneously destabilize multiple alternative pairings, studies were carried out on G11C/U27Delta in the presence of urea or increased monovalent ion concentration. Inclusion of physiological ionic strength allowed the goal of monophasic, fast-folding (kobs approximately 60 min(-1)) kinetics to be realized. To account for this, a model is developed wherein Na+, which destabilizes secondary and tertiary structures in the presence of Mg2+, facilitates native folding by destabilizing the multiple alternative secondary structures with a higher-order dependence.
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PMID:Design of a highly reactive HDV ribozyme sequence uncovers facilitation of RNA folding by alternative pairings and physiological ionic strength. 1528 80

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.
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PMID:Cations and hydration in catalytic RNA: molecular dynamics of the hepatitis delta virus ribozyme. 1661 77


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