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RNA. 2004 Jul;10(7):1059-72.

Cross-linking experiments reveal the presence of novel structural features between a hepatitis delta virus ribozyme and its substrate.

Author information

1
RNA group/groupe ARN, Département de biochimie, Faculté de médecine, Université de Sherbrooke, Québec J1H 5N4, Canada.

Abstract

The kinetic pathway of a trans-acting delta ribozyme includes an essential structural rearrangement involving the P1 stem, a stem that is formed between the substrate and the ribozyme. We performed cross-linking experiments to determine the substrate position within the catalytic center of an antigenomic, trans-acting, delta ribozyme. Substrates that included a 4-thiouridine either in position -1, +4, or +8 (i.e., adjacent to the cleavage site, or located either in the middle of or at the 3'-end of the P1 stem, respectively) were synthesized and shown to be efficiently cleaved. Examination of the cross-linking conditions, the use of various mutated ribozymes, as well as the probing and characterization of the resulting ribozyme-substrate complexes, revealed several new features of the molecular mechanism: (1) the close proximity of several bases between nucleotides of the substrate and ribozyme; (2) the active ribozyme-substrate complex folds in a manner that docks the middle of the P1 stem on the P3 stem, while concomitantly the scissile phosphate is in close proximity to the catalytic cytosine; and, (3) some complexes appear to be compatible with being active intermediates along the folding pathway, while others seem to correspond to misfolded structures. To provide a model representation of these data, a three-dimensional structure of the delta ribozyme was developed using several RNA bioinformatic software packages.

PMID:
15208442
PMCID:
PMC1370597
DOI:
10.1261/rna.7230604
[Indexed for MEDLINE]
Free PMC Article
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