Escherichia coli initiator tRNA: structure-function relationships and interactions with the translational machinery

We showed previously that the sequence and (or) structural elements important for specifying the many distinctive properties of Escherichia coli initiator tRNA are clustered in the acceptor stem and in the anticodon stem and loop. This paper briefly describes this and reviews the results of some recently published studies on the mutant initiator tRNAs generated during this work. First, we have studied the effect of overproduction of methionyl-tRNA transformylase (MTF) and initiation factors IF2 and IF3 on activity of mutant initiator tRNAs that are defective at specific steps in the initiation pathway. Overproduction of MTF rescued specifically the activity of mutant tRNAs defective in formylation but not mutants defective in binding to the P site. Overproduction of IF2 increased the activity of all mutant tRNAs having the CUA anticodon but not of mutant tRNA having the GAC anticodon. Overproduction of IF3 had no effect on the activity of any of the mutant tRNAs tested. Second, for functional studies of mutant initiator tRNA in vivo, we used a CAU --> CUA anticodon sequence mutant that can initiate protein synthesis from UAG instead of AUG. In contrast with the wild-type initiator tRNA, the mutant initiator tRNA has a 2-methylthio-N6-isopentenyl adenosine (ms2i6A) base modification next to the anticodon. Interestingly, this base modification is now important for activity of the mutant tRNA in initiation. In a miaA strain of E. coli deficient in biosynthesis of ms2i6A, the mutant initiator tRNA is much less active in initiation. The defect is specifically in binding to the ribosomal P site.