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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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Hypothetical representation of the folding of the J4/2 junction region before and after the formation of the P1.1 pseudoknot. (A) Nucleotide sequence and secondary structure of the antigenomic ribozyme characterized in this work. The substrate is represented by the thin line in order to simplify the representation. (B) 3D model representation of the antigenomic ribozyme drawn based on the backbone structure obtained from the crystal structure of the genomic version (3). The structural motifs are identified.
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Figure 8: Hypothetical representation of the folding of the J4/2 junction region before and after the formation of the P1.1 pseudoknot. (A) Nucleotide sequence and secondary structure of the antigenomic ribozyme characterized in this work. The substrate is represented by the thin line in order to simplify the representation. (B) 3D model representation of the antigenomic ribozyme drawn based on the backbone structure obtained from the crystal structure of the genomic version (3). The structural motifs are identified.

Mentions: The molecular switch leading to the formation of the C19–G80 bp appears to be one feature of a complex structural rearrangement that either occurred simultaneously with, or rapidly following, the formation of the P1.1 pseudoknot. The latter step, which was shown to take place after both the annealing of the substrate to the ribozyme and the docking of the P1 stem within the catalytic centre, has been proposed to be the limiting step of the HDV ribozyme catalysis (8,24). The folding of the P1.1 pseudoknot appears to be the central event that triggers all of the conformational changes leading to the cleavage reaction. This results in the complete folding of the J4/2 junction, including the formation of the C19–G80 bp, the ribose zipper (including the A78A79) and the trefoil turn (C76U77) (Figure 8). Together, the formation of these features leads to the correct positioning of the catalytic cytosine (C76). Furthermore, G81 is most likely extruded out of the structure, as is U77. The extrusion of U77 is associated with the positioning of C76 deeply within the catalytic centre (11). This structural rearrangement might be viewed as two opposing forces causing the exclusion of one nucleotide. In the case of G81, it is tempting to suggest that this is reminiscent of a trefoil turn. Prior to this study, the presence of the structural motifs that form the J4/2 junction, with the exception of the base pair switch C19–G80, were all discovered by high-resolution approaches (3,4,23–25). The present work provides an original biochemical study of these motifs, as well as attempting to determine their order of formation. Moreover, it shows that G81 is optional for the ribozyme's catalysis. A mutant lacking G81 and including either a C19–G80 or U19–A80 bp exhibited virtually the same cleavage activity as did the wild-type ribozyme. Therefore, the conservation of this guanosine in all natural antigenomic variants is intriguing. Similarly, the GG homopurine bp is perfectly conserved in all of the natural variants, while in vitro selection revealed that the most efficient homopurine bp is AA. In addition, we observed several guanosine residues outside of the catalytic centre of the ribozyme (e.g. in the P4 stem-loop) that were conserved in the natural variants, but are not critical to the self-cleavage activity. According to the secondary structure predicted for several HDV RNA genome variants, most, if not all, of these guanosines appear to form base pairs. Therefore, it is tempting to speculate that a potential contribution of these guanosines would explain their conservation in the natural variants and they favour both, the unfolding of the ribozyme after cleavage and the subsequent formation of the rod-like structure that prevents the self-cleavage of the newly circular HDV RNA strands. Clearly, verifying this hypothesis requires additional experimentation.Figure 8.


A novel structural rearrangement of hepatitis delta virus antigenomic ribozyme.

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

Hypothetical representation of the folding of the J4/2 junction region before and after the formation of the P1.1 pseudoknot. (A) Nucleotide sequence and secondary structure of the antigenomic ribozyme characterized in this work. The substrate is represented by the thin line in order to simplify the representation. (B) 3D model representation of the antigenomic ribozyme drawn based on the backbone structure obtained from the crystal structure of the genomic version (3). The structural motifs are identified.
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Related In: Results  -  Collection

License
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getmorefigures.php?uid=PMC2175327&req=5

Figure 8: Hypothetical representation of the folding of the J4/2 junction region before and after the formation of the P1.1 pseudoknot. (A) Nucleotide sequence and secondary structure of the antigenomic ribozyme characterized in this work. The substrate is represented by the thin line in order to simplify the representation. (B) 3D model representation of the antigenomic ribozyme drawn based on the backbone structure obtained from the crystal structure of the genomic version (3). The structural motifs are identified.
Mentions: The molecular switch leading to the formation of the C19–G80 bp appears to be one feature of a complex structural rearrangement that either occurred simultaneously with, or rapidly following, the formation of the P1.1 pseudoknot. The latter step, which was shown to take place after both the annealing of the substrate to the ribozyme and the docking of the P1 stem within the catalytic centre, has been proposed to be the limiting step of the HDV ribozyme catalysis (8,24). The folding of the P1.1 pseudoknot appears to be the central event that triggers all of the conformational changes leading to the cleavage reaction. This results in the complete folding of the J4/2 junction, including the formation of the C19–G80 bp, the ribose zipper (including the A78A79) and the trefoil turn (C76U77) (Figure 8). Together, the formation of these features leads to the correct positioning of the catalytic cytosine (C76). Furthermore, G81 is most likely extruded out of the structure, as is U77. The extrusion of U77 is associated with the positioning of C76 deeply within the catalytic centre (11). This structural rearrangement might be viewed as two opposing forces causing the exclusion of one nucleotide. In the case of G81, it is tempting to suggest that this is reminiscent of a trefoil turn. Prior to this study, the presence of the structural motifs that form the J4/2 junction, with the exception of the base pair switch C19–G80, were all discovered by high-resolution approaches (3,4,23–25). The present work provides an original biochemical study of these motifs, as well as attempting to determine their order of formation. Moreover, it shows that G81 is optional for the ribozyme's catalysis. A mutant lacking G81 and including either a C19–G80 or U19–A80 bp exhibited virtually the same cleavage activity as did the wild-type ribozyme. Therefore, the conservation of this guanosine in all natural antigenomic variants is intriguing. Similarly, the GG homopurine bp is perfectly conserved in all of the natural variants, while in vitro selection revealed that the most efficient homopurine bp is AA. In addition, we observed several guanosine residues outside of the catalytic centre of the ribozyme (e.g. in the P4 stem-loop) that were conserved in the natural variants, but are not critical to the self-cleavage activity. According to the secondary structure predicted for several HDV RNA genome variants, most, if not all, of these guanosines appear to form base pairs. Therefore, it is tempting to speculate that a potential contribution of these guanosines would explain their conservation in the natural variants and they favour both, the unfolding of the ribozyme after cleavage and the subsequent formation of the rod-like structure that prevents the self-cleavage of the newly circular HDV RNA strands. Clearly, verifying this hypothesis requires additional experimentation.Figure 8.

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