S-adenosylmethionine and tetrahydrofolate-dependent methylation of tRNA in Bacillus subtilis: Incomplete methylations caused by trimethoprim, pactamycin, or chloramphenicol

Abstract

The major methylated bases of tRNA of Bacillus subtilis are 5-methyluracil (m 5U), 7-methylguanine (m 7G) and 1-methyladenine (m 1A). In addition, tRNA from stationary phase cultures always contains considerable amounts of an unidentified methylated pyrimidine derivative, “P.” Trimethoprim, which inhibits tetrahydrofolate (FH 4)-dependent transmethylation reactions, was used to accumulate m 5U-deficient tRNA in B. subtilis. With Escherichia coli enzymes the methyl moiety of S-adenosylmethionine (SAM) can be incorporated into the tRNA, and 0.7 m 5U residue is formed per one tRNA molecule. The FH 4-dependent formation of m 5U in tRNA of B. subtilis as well as SAM-dependent methylations are affected by antibiotics which interfere with ribosomal functions. tRNA from pactamycin- or chloramphenicol-treated cells serves as substrate for SAM-dependent m 5U- and m 7G-specific tRNA methyltransferases from E. coli. With homologous SAM-dependent tRNA methyltransferases considerably fewer, but specific, transmethylations were found. tRNA obtained from a methionine-requiring stringent mutant of B. subtilis starved for methionine in the presence of chloramphenicol accepts the methyl moiety from SAM with homologous enzymes. The pattern of the methylated bases upon in vitro methylation of this tRNA agrees well with the pattern of methioninederived methylated bases in total tRNA of B. subtilis. Significant alterations were found in the relative distribution of tRNA isoacceptors from pactamycin-treated B. subtilis, which may reflect an accumulation of premature tRNAs.