Kinetic analysis of delta ribozyme cleavage.
The ability of delta ribozyme to catalyze the cleavage of an 11-mer RNA substrate was examined under both single- and multiple-turnover conditions. In both cases only small differences in the kinetic parameters were observed in the presence of either magnesium or calcium as cofactor. Under multiple-turnover conditions, the catalytic efficiency of the ribozyme (kcat/KM) was higher at 37 degreesC than at 56 degreesC. The cleavage reaction seems to be limited by the product release step at 37 degreesC and by the chemical cleavage step at 56 degreesC. We observed substrate inhibition at high concentrations of the 11-mer substrate. Cleavage rate constants were determined with a structural derivative characterized by an ultrastable L4 tetraloop. The kinetic parameters (kcat and KM) and dissociation constant (Kd) were almost identical for both ribozymes, suggesting that the stability of the L4 loop has a negligible impact on the catalytic activities of the examined ribozymes. Various cleavage inhibition and gel-shift assays with analogues, substrate, and both active and inactive ribozymes were performed. The 2'-hydroxyl group adjacent to the scissile phosphate was shown to be involved in binding with the ribozyme, while the essential cytosine residue of the J4/2 junction was shown to contribute to substrate association. We clearly show that substrate binding to the delta ribozyme is not restricted to the formation of a helix located downstream of the cleavage site. Using these results, we postulate a kinetic pathway involving a conformational transition step essential for the formation of the active ribozyme/substrate complex.
Mercure
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Département de biochimie, Faculté de médecine, Université de Sherbrooke, Sherbrooke, Québec J1H 5N4, Canada.
Lafontaine
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D
Ananvoranich
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Perreault
J P
JP
D002118:Calcium / Q000494:pharmacology; D003698:Hepatitis Delta Virus; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008956:Models, Chemical; D012323:RNA Processing, Post-Transcriptional; D016337:RNA, Catalytic / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D012367:RNA, Viral / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*
D016337:RNA, Catalytic; D012367:RNA, Viral; D008274:Magnesium; D002118:Calcium