IMPORTANT ROLE OF THE AMINO-ACID ATTACHED TO TRANSFER-RNA IN FORMYLATION AND IN INITIATION OF PROTEIN-SYNTHESIS IN ESCHERICHIA-COLI

In attempts to convert an elongator tRNA to an initiator tRNA, we prev iously generated a mutant elongator methionine tRNA carrying an antico don sequence change from CAU to CUA along with the two features import ant for activity of Escherichia coli initiator tRNA in initiation. Thi s mutant tRNA (Mi:2 tRNA) was active in initiation in vivo but only wh en aminoacylated with methionine by overproduction of methionyl-tRNA s ynthetase. Here we show that the Mi:2 tRNA is normally aminoacylated i n vivo with lysine and that the tRNA aminoacylated with lysine is a ve ry poor substrate for formylation compared with the same tRNA aminoacy lated with methionine. By introducing further changes at base pairs 4: 69 and 5:68 in the acceptor stem of the Mi:2 tRNA to those found in th e E. coli initiator tRNA, we show that change of the U4:A69 base pair to G4:C69 and overproduction of lysyl-tRNA synthetase and methionyl-tR NA transformylase results in partial formylation of the mutant tRNA an d activity of the formyllysyl-tRNAs in initiation of protein synthesis . Thus, the G4:C69 base pair contributes toward formylation of the tRN A and protein synthesis in E. coli can be initiated with formyllysine. We also discuss the implications of these and other results on recogn ition of tRNAs by E. coli lysyl-tRNA synthetase and on competition in cells among aminoacyl-tRNA synthetases.