Methylation and Processing of Transfer Ribonucleic Acid in Mammalian and Bacterial Cells

The relationship between the methylation and processing of tRNA in both bacterial and mammalian cell systems was investigated by assessing the methylation of an existing population of precursor-tRNAs in the absence of tRNA synthesis. When the synthesis of tRNA in Escherichia coli B (rifampicin) and human KB cells (actinomycin D) was inhibited with the appropriate antibiotic, the incorporation of [3H[methyl groups into tRNA (via [methyl-3H]methionine labeling) rapidly declined with time and was essentially complete within 30 and 60 min, respectively. Although antibiotic treatment predictably reduced the incorporation of methyl groups into tRNA, it also resulted in significant changes in the distribution of the type of methylated products formed. Thus, for KB cells the marked increases in the per cent of radioactivity incorporated into 2'-0-methylribose derivatives, N2-methylguanine, and 3-methylcytosine of tRNA preparation pre-chased with actinomycin D for progressively longer periods of time prior to labeling with [methyl-3H]methionine led to the interpretation that these methylated constituents were formed predominantly during the late stages of tRNA maturation. Similarly, progessive and marked decreases in 1-, 7-, and N2, N2-methylguanine, and moderate decreases in 1-methyladenosine, 5-methylcytosine, and 5-methyluracil revealed that these methylated products were formed primarily during the early and intermediate stages of maturation, respectively. Similar analysis of E. coli B methylation products indicated that the bulk of methyl groups incorporated into the base moieties of tRNA (1- and 7-methylguanine, 2- and N6-methyladenine, and 5-methyluracil) occurred prior to the formation of 2'-0-methylribose derivatives. Additional evidence is presented which negates the possibility that an ancillary action of these antibiotics was the inhibition of specific tRNA-methyl-transferase enzymes.