Aminoacylation of undermethylated mammalian transfer RNA.
To study the role of 5-methylcytidine in the aminoacylation of mammalian tRNA, bulk tRNA specifically deficient in 5-methylcytidine was isolated from the livers of mice treated with 5-azacytidine (18 mg/kg) for 4 days. For comparison, more extensively altered tRNA was isolated from the livers of mice treated with DL-ethionine (100 mg/kg) plus adenine (48 mg/kg) for 3 days. The amino acid acceptor capacity of these tRNAs was determined by measuring the incorporation of one of eight different 14C-labeled amino acids or a mixture of 14C-labeled amino acids in homologous assays using a crude synthetase preparation isolated from untreated mice. The 5-methylcytidine-deficient tRNA incorporated each amino acid to the same extent as fully methylated tRNA. The tRNA from DL-ethionine-treated livers showed an overall decreased amino-acylation capacity for all amino acids tested. The 5-methylcytidine-deficient tRNA from DL-ethionine-treated mice were further characterized as substrates in homologous rate assays designed to determine the Km and V of the aminoacylation reaction using four individual 14C-labeled amino acids and a mixture of 14C-labeled amino acids. The Km and V of the reactions for all amino acids tested using 5-methylcytidine-deficient tRNA as substrate were essentially the same as for fully methylated tRNA. However, the Km and V were increased when liver tRNA from mice treated with DL-ethionine plus adenine was used as substrate in the rate reaction with [14C]lysine as label. Our results suggest that although extensively altered tRNA is a poorer substrate than control tRNA in both extent and rate of aminoacylation, 5-methylcytidine in mammalian tRNA is not involved in the recognition of the tRNA by the synthetase as measured by aminoacylation activity.
D000596:Amino Acids / Q000378:metabolism; D000604:Amino Acyl-tRNA Synthetases / Q000378:metabolism*; D000818:Animals; D001374:Azacitidine / Q000494:pharmacology; D005001:Ethionine / Q000494:pharmacology; D007700:Kinetics; D008099:Liver / Q000187:drug effects / Q000201:enzymology*; D008297:Male; D051379:Mice; D012343:RNA, Transfer / Q000378:metabolism*; D012346:RNA, Transfer, Amino Acyl / Q000096:biosynthesis*
D000596:Amino Acids; D012346:RNA, Transfer, Amino Acyl; D012343:RNA, Transfer; D000604:Amino Acyl-tRNA Synthetases; D001374:Azacitidine; D005001:Ethionine