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J. Biol. Chem., Vol. 277, Issue 29, 26508-26516, July 19, 2002

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Kinetic and Binding Analysis of the Catalytic Involvement of Ribose Moieties of a trans-Acting  Ribozyme^*

Karine Fiola and Jean-Pierre Perreault^§

From the RNA Group/Groupe ARN, Département de Biochimie, Université de Sherbrooke, Sherbrooke, Québec J1H 5N4, Canada

We have identified ribose 2'-hydroxyl groups (2'-OHs) that are critical for the activity of a trans-cleaving  ribozyme derived from the antigenomic strand of the hepatitis  virus. Initially, an RNA-DNA mixed ribozyme composed of 26 deoxyribo- (specifically the nucleotides forming the P2 stem and the P4 stem-loop) and 31 ribonucleotides (those forming the catalytic center) was engineered. This mixed ribozyme catalyzed the cleavage of a small substrate with kinetic parameters virtually identical to those of the all-RNA ribozyme. The further substitution of deoxyribose for ribose residues permitted us to investigate the contribution of all 2'-OHs to catalysis. Determination of the kinetic parameters for the cleavage reaction of the resulting ribozymes revealed (i) 10 2'-OH groups appear to be important in supporting the formation of several hydrogen bonds within the catalytic core, (ii) none of the important 2'-OHs seem to coordinate a magnesium cation, and (iii) 1 of the tested RNA-DNA mixed polymers appeared to stabilize the ribozyme-substrate transition-state complex, resulting in an improvement over the all-RNA counterpart. The contribution of the 2'-OHs to the catalytic mechanism is discussed, and differences with the crystal structure of a genomic  self-cleaved product are explained. Clearly, the 2'-OHs are essential components of the network of interactions involved in the formation of the catalytic center of the  ribozyme.

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