RECOGNITION OF THE T-ARM OF TRANSFER-RNA BY TRANSFER-RNA (M(5)U54)-METHYLTRANSFERASE IS NOT SEQUENCE-SPECIFIC

tRNA (m(5)U54)-methyltransferase (RUMT) catalyzes the methylation of U 54 of tRNAs. In contrast to enzymes which recognize a particular tRNA, RUMT recognizes features common to all tRNAs. We have shown that thes e features reside in the T-arm of tRNA and constructed a minimal conse nsus sequence for RUMT recognition and catalysis (Gu et al., 1991b). H ere, we have mutated each conserved T-loop residue and conserved T-ste m base pair to bases or base pairs which are net observed in Escherich ia coli tRNA. The substrate specificity of RUMT for 30 in vitro synthe sized T-arm mutants of tRNA(Phe) and 37 mutants of the 17-mer analog o f the T-arm derived from tRNA(1)(Val) was investigated, A 2-5 base pai r stem was essential for recognition of the T-arm by RUMT, but the bas e composition of the stem was unimportant. The 7-base size of the T-lo op maintained by the stem was essential for RUMT recognition. For tRNA , most base substitutions in the 7-base loop did not eliminate RUMT ac tivity, except for any mutation of the methyl acceptor U54 and the C56 G mutation. The effect of base and base pair mutations on k(cat) or th e rate of methylation by RUMT was more striking than the effect on the K-d for binding to RUMT. In comparison with mutations in the T-loop o f intact tRNA, base mutation in the T-loop of the 17-mer T-arm had a m ore deleterious effect on binding and methylation. Surprisingly, recog nition of tRNA by RUMT appears to reside in the three-dimensional stru cture of the seven-member T-loop rather than in its primary structure.