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

Karine Fiola,Jean-Pierre Perreault

doi:10.1074/jbc.m203468200

Abstract

We have identified ribose 2'-hydroxyl groups (2'-OHs) that are critical for the activity of a trans-cleaving delta ribozyme derived from the antigenomic strand of the hepatitis delta 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 delta 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 delta ribozyme.

Highlights

Both genomic and antigenomic hepatitis ␦ virus RNAs exhibit self-cleavage activity, a process involved in viral replication
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
RNA strands, the first step of this project was the development of a version of ␦ ribozyme containing fewer ribonucleotides

Summary

The abbreviations used are

Ribozyme; 2Ј-H, 2Ј-hydrogen atom; 2Ј-OH, 2Ј-hydroxyl group; nt, nucleotide(s); ␦RD-Rz, ␦RNA-DNA ribozyme; S, substrate. It has been demonstrated that imidazole buffer rescues the activity of a mutant antigenomic-derived ␦ ribozyme possessing U76 instead of the usual C76 (referred as C47 in the transacting ␦ ribozyme used here) [5] This result suggests that C76 acts as a general base in the catalytic mechanism. We use an RNA-DNA mixed ribozyme with P2 and P4 stems, which surround the catalytic center, composed exclusively of deoxyribonucleotides except for one base pair in each stem Because this RNA-DNA mixed ribozyme has kinetic parameters virtually identical to those of the all-RNA version, we used this new tool to identify all 2Ј-OHs important in supporting the cleavage activity of the trans-acting ␦ ribozyme

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION