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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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Cleavage activity assays of various mutants of the J4/2 junction. The inset shows a typical autoradiogram of a PAGE gel for a 30 min reaction for each ribozyme tested. The secondary structure of the wild-type ribozyme is illustrated entirely on the left, while only the P2 stem, J4/2 junction and the most relevant nucleotides are illustrated for the mutants. The mutations and insertion are denoted by squared and circled nucleotides, respectively, while deletions are indicated by a triangle (▵). The lane numbers at the top of the autoradiogram correspond to the number of each ribozyme: 1, wild-type; 2, RzA78U,A79U; 3, Rz–U+79–80; 4, Rz–C19A,G81U,G80A; 5, Rz–U+76–77; 6, Rz–▵77; 7 is the original bimolecular construct (RzA–RzB); and, 8, the bimolecular construct including an abasic residue (Ab) in position 77 (RzB–Ab77). The positions of the substrate and product are indicated adjacent to the gel. The rate constants (kobs) for each ribozyme are indicated below the gel.
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Figure 6: Cleavage activity assays of various mutants of the J4/2 junction. The inset shows a typical autoradiogram of a PAGE gel for a 30 min reaction for each ribozyme tested. The secondary structure of the wild-type ribozyme is illustrated entirely on the left, while only the P2 stem, J4/2 junction and the most relevant nucleotides are illustrated for the mutants. The mutations and insertion are denoted by squared and circled nucleotides, respectively, while deletions are indicated by a triangle (▵). The lane numbers at the top of the autoradiogram correspond to the number of each ribozyme: 1, wild-type; 2, RzA78U,A79U; 3, Rz–U+79–80; 4, Rz–C19A,G81U,G80A; 5, Rz–U+76–77; 6, Rz–▵77; 7 is the original bimolecular construct (RzA–RzB); and, 8, the bimolecular construct including an abasic residue (Ab) in position 77 (RzB–Ab77). The positions of the substrate and product are indicated adjacent to the gel. The rate constants (kobs) for each ribozyme are indicated below the gel.

Mentions: The results described above demonstrate that initially C19 is base paired to G81 and either simultaneously with, or after the formation of, the P1.1 pseudoknot this base pair is disrupted in order to permit formation of the C19–G80 bp (Figure 4A). The formation of the P1.1 pseudoknot has been demonstrated to be the limiting step of the HDV folding pathway, in addition to being critical for the folding of the complete J4/2 junction (3,8). The folding of the J4/2 junction involves the formation of a ribose zipper, a trefoil turn and the positioning of the catalytic cytosine within the catalytic core (C76), three features that received physical support from X-ray diffraction, nuclear magnetic resonance (NMR) and fluorescence spectroscopy studies (3,4,23–25). In order to acquire additional biochemical understanding of the J4/2 junction's folding, we analysed our sequence database in terms of these structural features and then synthesized mutated trans-acting ribozymes in order to evaluate their cleavage activities (Figure 6).Figure 6.


A novel structural rearrangement of hepatitis delta virus antigenomic ribozyme.

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

Cleavage activity assays of various mutants of the J4/2 junction. The inset shows a typical autoradiogram of a PAGE gel for a 30 min reaction for each ribozyme tested. The secondary structure of the wild-type ribozyme is illustrated entirely on the left, while only the P2 stem, J4/2 junction and the most relevant nucleotides are illustrated for the mutants. The mutations and insertion are denoted by squared and circled nucleotides, respectively, while deletions are indicated by a triangle (▵). The lane numbers at the top of the autoradiogram correspond to the number of each ribozyme: 1, wild-type; 2, RzA78U,A79U; 3, Rz–U+79–80; 4, Rz–C19A,G81U,G80A; 5, Rz–U+76–77; 6, Rz–▵77; 7 is the original bimolecular construct (RzA–RzB); and, 8, the bimolecular construct including an abasic residue (Ab) in position 77 (RzB–Ab77). The positions of the substrate and product are indicated adjacent to the gel. The rate constants (kobs) for each ribozyme are indicated below the gel.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 6: Cleavage activity assays of various mutants of the J4/2 junction. The inset shows a typical autoradiogram of a PAGE gel for a 30 min reaction for each ribozyme tested. The secondary structure of the wild-type ribozyme is illustrated entirely on the left, while only the P2 stem, J4/2 junction and the most relevant nucleotides are illustrated for the mutants. The mutations and insertion are denoted by squared and circled nucleotides, respectively, while deletions are indicated by a triangle (▵). The lane numbers at the top of the autoradiogram correspond to the number of each ribozyme: 1, wild-type; 2, RzA78U,A79U; 3, Rz–U+79–80; 4, Rz–C19A,G81U,G80A; 5, Rz–U+76–77; 6, Rz–▵77; 7 is the original bimolecular construct (RzA–RzB); and, 8, the bimolecular construct including an abasic residue (Ab) in position 77 (RzB–Ab77). The positions of the substrate and product are indicated adjacent to the gel. The rate constants (kobs) for each ribozyme are indicated below the gel.
Mentions: The results described above demonstrate that initially C19 is base paired to G81 and either simultaneously with, or after the formation of, the P1.1 pseudoknot this base pair is disrupted in order to permit formation of the C19–G80 bp (Figure 4A). The formation of the P1.1 pseudoknot has been demonstrated to be the limiting step of the HDV folding pathway, in addition to being critical for the folding of the complete J4/2 junction (3,8). The folding of the J4/2 junction involves the formation of a ribose zipper, a trefoil turn and the positioning of the catalytic cytosine within the catalytic core (C76), three features that received physical support from X-ray diffraction, nuclear magnetic resonance (NMR) and fluorescence spectroscopy studies (3,4,23–25). In order to acquire additional biochemical understanding of the J4/2 junction's folding, we analysed our sequence database in terms of these structural features and then synthesized mutated trans-acting ribozymes in order to evaluate their cleavage activities (Figure 6).Figure 6.

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