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A novel structural rearrangement of hepatitis delta virus antigenomic ribozyme.

Nehdi A, Perreault J, Beaudoin JD, Perreault JP - Nucleic Acids Res. (2007)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: RNA Group/Groupe ARN, Département de Biochimie, Faculté de médecine et des sciences de la santé, Université de Sherbrooke, Sherbrooke, Québec, J1H 5N4, Canada.

ABSTRACT
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.

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Related in: MedlinePlus

Histogram of the determined rate constant (kobs) for the wild-type ribozyme (1), the RzG81A (2), the RzG81C (3) and the RzG41U and (4) mutants.
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Figure 3: Histogram of the determined rate constant (kobs) for the wild-type ribozyme (1), the RzG81A (2), the RzG81C (3) and the RzG41U and (4) mutants.

Mentions: The base pairing of the nucleotides at the bottom of the P2 stem (C19–G81) was believed to form a Watson–Crick bp (Figure 1). The covariation analysis does not support the presence of this base pair (see Table 1 and Supplementary data Table 2). According to the set of selected sequences, only 19% of the selected variants possess either a Watson–Crick or Wobble bp at this position (Table 1). CSs attributed to the different nucleotides that can form Watson–Crick bp are either very close to zero, or negative (e.g. A19 − U81 = 0; U19 − A81 = − 0.2; G19 − C81 = − 0.1; C19 − G81 = 0.08). In order to add biochemical support to this observation, trans-acting mutant ribozymes, including the three possible mutations at position 81, were synthesized and their cleavage activities assessed under single-turnover conditions. The rate constants (kobs) of the mutants were determined, and are presented in histogram form (Figure 3). The least active mutant was the wild-type ribozyme harbouring a C19–G81 bp. The three mutants that did not permit the formation of a base pair were all more active. Independent experiments with several other mutants at the bottom of the P2 stem (positions 19 and 81) also led to the same conclusion. Even several ribozymes without a base pair at this position were found to be more active than the wild-type ribozyme (Ouellet,J. and Perreault,J.P, unpublished data).Figure 3.


A novel structural rearrangement of hepatitis delta virus antigenomic ribozyme.

Nehdi A, Perreault J, Beaudoin JD, Perreault JP - Nucleic Acids Res. (2007)

Histogram of the determined rate constant (kobs) for the wild-type ribozyme (1), the RzG81A (2), the RzG81C (3) and the RzG41U and (4) mutants.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2175327&req=5

Figure 3: Histogram of the determined rate constant (kobs) for the wild-type ribozyme (1), the RzG81A (2), the RzG81C (3) and the RzG41U and (4) mutants.
Mentions: The base pairing of the nucleotides at the bottom of the P2 stem (C19–G81) was believed to form a Watson–Crick bp (Figure 1). The covariation analysis does not support the presence of this base pair (see Table 1 and Supplementary data Table 2). According to the set of selected sequences, only 19% of the selected variants possess either a Watson–Crick or Wobble bp at this position (Table 1). CSs attributed to the different nucleotides that can form Watson–Crick bp are either very close to zero, or negative (e.g. A19 − U81 = 0; U19 − A81 = − 0.2; G19 − C81 = − 0.1; C19 − G81 = 0.08). In order to add biochemical support to this observation, trans-acting mutant ribozymes, including the three possible mutations at position 81, were synthesized and their cleavage activities assessed under single-turnover conditions. The rate constants (kobs) of the mutants were determined, and are presented in histogram form (Figure 3). The least active mutant was the wild-type ribozyme harbouring a C19–G81 bp. The three mutants that did not permit the formation of a base pair were all more active. Independent experiments with several other mutants at the bottom of the P2 stem (positions 19 and 81) also led to the same conclusion. Even several ribozymes without a base pair at this position were found to be more active than the wild-type ribozyme (Ouellet,J. and Perreault,J.P, unpublished data).Figure 3.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: RNA Group/Groupe ARN, Département de Biochimie, Faculté de médecine et des sciences de la santé, Université de Sherbrooke, Sherbrooke, Québec, J1H 5N4, Canada.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus