Recognition of the T-arm of tRNA by tRNA (m5U54)-methyltransferase is not sequence specific.

tRNA (m5U54)-methyltransferase (RUMT) catalyzes the methylation of U54 of tRNAs. In contrast to enzymes which recognize a particular tRNA, RUMT recognizes features common to all tRNAs. We have shown that these features reside in the T-arm of tRNA and constructed a minimal consensus sequence for RUMT recognition and catalysis (Gu et al., 1991b). Here, we have mutated each conserved T-loop residue and conserved T-stem base pair to bases or base pairs which are not observed in Escherichia coli tRNA. The substrate specificity of RUMT for 30 in vitro synthesized T-arm mutants of tRNAPhe and 37 mutants of the 17-mer analog of the T-arm derived from tRNA1Val was investigated. A 2-5 base pair stem was essential for recognition of the T-arm by RUMT, but the base composition of the stem was unimportant. The 7-base size of the T-loop maintained by the stem was essential for RUMT recognition. For tRNA, most base substitutions in the 7-base loop did not eliminate RUMT activity, except for any mutation of the methyl acceptor U54 and the C56G mutation. The effect of base and base pair mutations on Kcat or the rate of methylation by RUMT was more striking than the effect on the Kd for binding to RUMT. In comparison with mutations in the T-loop of intact tRNA, base mutation in the T-loop of the 17-mer T-arm had a more deleterious effect on binding and methylation. Surprisingly, recognition of tRNA by RUMT appears to reside in the three-dimensional structure of the seven-member T-loop rather than in its primary structure.