IN-VITRO METHYLATION OF ESCHERICHIA-COLI 16S RIBOSOMAL-RNA BY TRANSFER-RNA (M(5)U54)-METHYLTRANSFERASE

16S rRNA, isolated from Escherichia coli or synthesized in vitro, is m ethylated by tRNA (m(5)U54)-methyltransferase (RUMT) and S-adenosyl-L- methionine to give ribothymidine (m(5)U). By methylation studies of 16 S rRNA fragments, nearest-neighbor analysis, and nuclease protection e xperiments, the site of methylation was identified as U788. We have pr eviously shown that the substrate consensus sequence for the T-arm of tRNA consists of a 2-5 base-pair stem and a 7-base loop, with certain constraints on base substitutions within the loop, and in the first tw o bases which close the loop [Gu, X., and Santi, D. V. (1991) Biochemi stry 30, 2999-3002]. U788 of 16S rNA is within a 9-base loop of a pred icted stem-loop structure of 16S rRNA. If Ado substitution is allowed at the third and seventh positions of the loop and the first and ninth bases of the loop form an A-C base pair, the resulting stem-loop fall s within the RUMT consensus sequence of the T-arm of tRNA. Individual mutants of the tRNA T-arm at these positions confirm that the substitu tions are allowable, and expand the previous consensus sequence. Furth er, prevention of 7-base loop formation by requiring C-C base-pair for mation at the loop closure abolishes substrate activity. RUMT forms a complex with Syn 16S rRNA which can be isolated on nitrocellulose filt ers or by SDS-PAGE electrophoresis. The enzyme also catalyzes exchange of tritium of [H-3]Ura-16S rRNA for protons of water. By analogy with studies with tRNA [Gu, X., and Santi, D. V. (1991) Biochemistry 31, 1 0295-10302], the mechanism of methylation is proposed to involve forma tion of a covalent, albeit reversible, Michael adduct with the target U788 of 16S rRNA.