Systematic substitution of individual bases in two important single‐stranded regions of the HDV ribozyme for evaluation of the role of specific bases

Suh, Young‐Ah; Kumar, Penmetcha K. R.; Kawakami, Junji; Nishikawa, Fumiko; Taira, Kazunari; Nishikawa, Satoshi

doi:10.1016/0014-5793(93)81782-u

Cited by 24 publications

(19 citation statements)

References 22 publications

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“…In the antigenomic ribozyme, an A78C mutation reduced self-cleavage activity by approximately the same amount [58]. With the genomic ribozyme, where the extent of cleavage during transcription, but not the rate of the reaction, was followed for all three substitutions at this position, only the A77G mutant cleaved to completion [56]. A small or moderate decrease in the rate of the reaction may not have been detected in those experiments, however.…”

Section: Variation Within the Hdv Ribozymesmentioning

confidence: 83%

Ribozyme activity in the genomic and antigenomic RNA strands of hepatitis delta virus

Wadkins¹,

Been²

2002

Cellular and Molecular Life Sciences (CMLS)

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In the hepatitis delta virus, ribozymes are encoded in both the genomic strand RNA and its complement, the antigenomic strand. The two ribozymes are similar in sequence and structure, are most active in the presence of divalent cation and catalyze RNA cleavage reactions which generate a 5'-hydroxyl group and a 2',3'-cyclic phosphate group. Recent progress has been made in understanding the catalytic mechanism. One key was a crystal structure of the genomic ribozyme that revealed a specific cytosine positioned to act as a general acid-base catalyst. The folding of the ribozyme in the context of the longer viral RNA is another area of interest. The biology requires that each ribozyme act only once, and mechanisms proposed for regulation of ribozyme activity sometimes invoke alternative RNA structures. Likewise, interference of ribozyme function by polyadenylation of the antigenomic RNA strand could be controlled through alternative structures, and a model for such control is proposed.

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Section: Variation Within the Hdv Ribozymesmentioning

confidence: 83%

Ribozyme activity in the genomic and antigenomic RNA strands of hepatitis delta virus

Wadkins¹,

Been²

2002

Cellular and Molecular Life Sciences (CMLS)

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“…C75 has been shown to play a direct role in catalysis, 14,16 and the mutation of C75 to U or G effectively eliminates ribozyme activity. [82][83][84][85] Mutation of C75 to adenosine lowers the apparent pK a of the reaction by an amount that corresponds to the difference in the solution pK a values of cytidine and adenosine, suggesting strongly that the apparent pK a of the reaction reflects that of the nucleotide at this position. 14,18 It has been proposed that the catalytic activity of HDVR depends on the protonation of C75.…”

Section: Identification Of Pk a -Shifted Nucleotides In Pseudoknotsmentioning

confidence: 94%

Calculation of pKas in RNA: On the Structural Origins and Functional Roles of Protonated Nucleotides

Tang

Alexov

Pyle

et al. 2007

Journal of Molecular Biology

134

175

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“…An ribozyme (7 pM) and substrate (14 pM) ratio of 1 : 2 was used for the second and third cycles; this ratio was kept almost constant up to generation 9 and, finally, the ratio was further lowered to 1 : 1 .5 (ribozyme, 2 pM, substrate, 3 pM). From the second to fifth selection cycle, the concentration of Mg" was reduced to 10 mM; this concentration was further decreased to 5 mM in the remaining selection cycle (generations [6][7][8][9][10][11][12][13][14]. After five cycles of selection, the reaction time for the ribozyme cleavage was also reduced from 1 h to 30 min in the successive cycles (generations 5 -14).…”

Section: Methodsmentioning

confidence: 99%

Selection in vitro of Trans ‐Acting Genomic Human Hepatitis Delta Virus (HDV) Ribozymes

Nishikawa¹,

Kawakami

Chiba

et al. 1996

European Journal of Biochemistry

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In an effort to identify the functional structure as well as new active variants of the trans-acting genomic ribozyine of human hepatitis delta virus (HDV), we applied an in vitro selection procedure. A total of 14 rounds of selection and amplification was repeated and various mutant ribozymes in GI0 and G14 pools analyzed. Active ribozymes which were isolated in the present study (from GI0 and G14) all possessed conserved bases (that were identified earlier) in the cis-acting molecule. A dominant clone G10-68 variant was accumulated in generation 14. Interestingly, when base substitutions were analyzed in G10-68 variant, we found that this variant appears to be close to antigenome-like HDV ribozyme molecule. Further investigations of G10-68 confirmed that each mutated base was the most appropriate nucleotide at every position of the HDV ribozyme.Keywords: human hepatitis D virus ; ribozyme ; in vitro selection ; pseudoknot structure ; PCR.Human hepatitis delta virus (HDV) is known to cause fulminant hepatitis or chronic hepatitis with linear cirrhosis due to hepatitis B virus [I]. The genome, a single-stranded circular RNA of about 1700 nucleotides, appears to replicate through a rolling circle RNA-to-RNA pathway as do some plant pathogenic RNA viruses [2]. Both genomic and antigenomic HDV RNAs have self-cleavage activities (ribozyme activity) in the presence of divalent metal ions ; they produce a 2',3'-cyclic phosphate and a 5'-0H group [2-41, as is the case for other known ribozymes, such as hammerhead and hairpin-type RNAs. However, the primary structure does not resemble those of the latter two types and, therefore, the HDV ribozyme represents an unique type of self-cleaving RNA.Earlier, secondary structural models were proposed for genomic and anti-genomic HDV ribozymes [4-81. In an attempt to understand the role of bases and to define the catalytic core structure of the genomic HDV ribozyme, we constructed several point and random variants in single stranded regions and also at stem regions [9-131. Our chemical probing studies [14] together with the above mutations, results support the pseudoknot secondary structure model. Recently, a tertiary structure model of the HDV ribozyme has also been described based on the pseudoknot structure [ 151 and phosphorothioate substitution interference analysis [ 161 supports this tertiary structure model. The cis-acting HDV ribozyme has been truncated to the transacting form by dividing cis-acting molecule into substrate and ribozyme portions 17, 17, 181.In order to isolate a new ribozymes or to improve the catalytic power or alter the substrate specificity for the ribozymes, the in vitro selection strategy has been widely used [19-261. We previously applied a simplified in vitro selection procedure to identify essential residues at one of the single-stranded regions of cis-acting HDV ribozyme [13]. This procedure was convenient for the identification of important residues but not suitable for isolating active variants of HDV ribozymes. In the present study, using a p...

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Systematic substitution of individual bases in two important single‐stranded regions of the HDV ribozyme for evaluation of the role of specific bases

Cited by 24 publications

References 22 publications

Ribozyme activity in the genomic and antigenomic RNA strands of hepatitis delta virus

Ribozyme activity in the genomic and antigenomic RNA strands of hepatitis delta virus

Calculation of pKas in RNA: On the Structural Origins and Functional Roles of Protonated Nucleotides

Selection in vitro of Trans ‐Acting Genomic Human Hepatitis Delta Virus (HDV) Ribozymes

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