Characterization of two species of methionine transfer ribonucleic acid from bakers' yeast.

The biochemical properties of two species of methionine transfer ribonucleic acid from bakers’ yeast, separated by diethylaminoethyl Sephadex column chromatography, have been studied. Each species of the two methionine tRNAs, designated methionine tRNA I and methionine tRNA II, respectively, has been purified by benzoylated DEAEcellulose column chromatography. Methionine tRNA I can be esterified with methionine by Escherichio coli aminoacyl-tRNA synthetase as efficiently as by yeast aminoacyl-tRNA synthetase, whereas methionine tRNA II can be charged only partially by the E. coli enzyme under the same conditions used in the charging of methionine tRNA I. Methionine tRNA I has been shown to be converted to N-formyhnethionyl-tRNA I by E. coli methionyltRNA transformylase in the presence of PO-formyltetrahydrofolate, while methionine tRNA1Iis-not,In-ribosomal binding studies, 14C-methionyl-tRNA I is recognized by the condons AUG, GUG, and UUG, while 14C-methionyl-tRNA II responds principally to AUG. In addition, methionine has been found to be incorporated from both methionyltRNAs I and II into polypeptide as directed by messenger RNA obtained from bacteriophage f2 in a cell-free system from E. coli. Our present report also provides evidence that yeast N-formyhnethionyl-tRNA I serves as an initiator of protein synthesis programmed with the natural messenger in an E. coli system in vitro. group from NlO-formyltetrahydrofolate, while the other (methionyl-tRNA& was not (2, 3). The specificity of these two methionyl-tRNAs in codon recognition has also been established by ribosomal binding and amino acid incorporation studies (24). Furthermore, N-formylmethionyl-tRNAF has been shown to serve as an initiator in polypeptide synthesis by a number of experiments utilizing an E. coli cell-free system, in which viral RNA (5-9) or synthetic polynucleotides (2, 4, 9-12) were used as messengers. It has also been confirmed that the same initiator participates in the protein synthesis directed by each cistron of a polycistronic messenger (13, 14). Recently, it has been shown, with natural messenger, that this is also the case with a system in vitro from Euglena gracilis (15). Little is known, however, about yeast methionine tRNA. It was previously shown that the methionine-accepting species of yeast tRNA was heterogeneous, since only a part of yeast methionine tRNA could be charged with E. coli aminoacyl-tRNA synthetase (16). However, further isolation and characterization of these tRNAs were not carried out. No definitive demonstration of methionyl-tRNA transformylase and of the involvement of N-formylmethionine in the chain initiation of protein synthesis in yeast has been encountered yet, although Marcker and Sanger (1) suggested the existence of N-formylmethionyl-tRNA in tRNA isolated from yeast grown in a medium containing V-labeled sulfate.