Mutants of Escherichia coli initiator tRNA defective in initiation. Effects of overproduction of methionyl-tRNA transformylase and the initiation factors IF2 and IF3

Abstract

We describe the effects of overproduction of methionyl-tRNA transformylase and initiation factors IF2 and IF3 on the activity, in vivo, of initiator tRNA mutants defective at specific steps of the initiation process in protein synthesis. The activity of the U35A36/G72 and U35A36/G72G73 mutants, which are defective in formylation, was increased by overproduction of methionyl-tRNA transformylase. In contrast, the activity of the C30:G40/U35A36 mutant, which is formylated normally but is defective in binding to the ribosomal P site, was not increased. Overproduction of IF2 had a strong stimulatory effect on the activity of virtually all the mutants carrying the U35A36 anticodon sequence change, including the U35A36, U35A36/G72, U35A36/G72G73, and the C30:G40/U35A36 mutants. In cells overproducing IF2, the amount of protein made by translation of a mutant mRNA, which uses the U35A36 mutant initiator tRNA, is severalfold higher than that made by translation of a wild type mRNA. We discuss the possible implications of this result on overproduction of proteins and on the order of assembly of the 30 S ribosome.mRNA.fMet-tRNA initiation complex in Escherichia coli. Over-production of IF3 did not affect the initiator activity of any of the tRNA mutants studied.