Recognition of the 5' leader and the acceptor stem of a pre-tRNA substrate by the ribozyme from Bacillus subtilis RNase P.
The catalysis by the ribozyme from bacterial RNase P involves specific interactions with the structure of the tRNA substrate. Recognition of the T stem-loop by this ribozyme occurs in a groove-like structure dictated by the tertiary folding of tRNA [Loria, A., and Pan, T. (1997) Biochemistry 36, 6317]. Effects of 2'-OH --> 2'-H modifications within the acceptor stem and the 5' leader on substrate binding and catalysis are determined using a tRNAPhe substrate that is significantly cleaved at more than one site. In all but one case, the 2'-deoxy substitution has little effect on binding for cleavage at the correct and incorrect sites. Substitution of the 2'-OH group at the correct site, however, decreases the cleavage chemistry by more than 3.4 kcal/mol for cleavage at both the correct and incorrect sites. Substitutions of the 2'-OH groups at the incorrect sites have no effect for cleavage at the incorrect and correct sites. Truncation of the 5' leader results in differential effects on cleavage at different sites. These observations lead to a model in which cleavage at the correct and incorrect sites involves formation of different ribozyme-substrate complexes depending on binding of specific nucleotides in the 5' leader. Binding of the T stem-loop of tRNA and the 2'-OH group at the correct cleavage site is common for all ES complexes. An A/U-rich 5' leader significantly promotes formation of the ES complex and accelerates the cleavage chemistry over those of a C/G-rich 5' leader, but only moderately enhances cleavage at the correct site over cleavage at the incorrect sites. Since cleavage at different sites requires formation of different ES complexes, cleavage site selection can occur at the level of the ES complex and at the chemical step.
Loria
A
A
Department of Biochemistry & Molecular Biology, University of Chicago, Illinois 60637, USA.
Pan
T
T
D001412:Bacillus subtilis / Q000201:enzymology*; D001482:Base Composition / Q000235:genetics; D001483:Base Sequence; D004722:Endoribonucleases / Q000378:metabolism*; D006868:Hydrolysis; D017665:Hydroxyl Radical; D016297:Mutagenesis, Site-Directed; D011522:Protons; D012322:RNA Precursors / Q000235:genetics / Q000378:metabolism*; D012329:RNA, Bacterial / Q000378:metabolism; D016337:RNA, Catalytic / Q000378:metabolism*; D012360:RNA, Transfer, Phe / Q000235:genetics / Q000378:metabolism*; D043262:Ribonuclease P; D013379:Substrate Specificity / Q000235:genetics
D011522:Protons; D012322:RNA Precursors; D012329:RNA, Bacterial; D016337:RNA, Catalytic; D012360:RNA, Transfer, Phe; D017665:Hydroxyl Radical; D004722:Endoribonucleases; D043262:Ribonuclease P