Kinetic and binding analysis of the catalytic involvement of ribose moieties of a trans-acting delta ribozyme.
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.
Fiola
Karine
K
RNA Group/Groupe ARN, Département de Biochimie, Université de Sherbrooke, Sherbrooke, Québec J1H 5N4, Canada.
Perreault
Jean-Pierre
JP
D001483:Base Sequence; D002384:Catalysis; D003698:Hepatitis Delta Virus / Q000235:genetics*; D006878:Hydroxides; D007700:Kinetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D016337:RNA, Catalytic / Q000737:chemistry*; D012367:RNA, Viral / Q000737:chemistry*
D006878:Hydroxides; D016337:RNA, Catalytic; D012367:RNA, Viral; C031356:hydroxide ion