ESCHERICHIA-COLI INITIATOR TRANSFER-RNA - STRUCTURE-FUNCTION-RELATIONSHIPS AND INTERACTIONS WITH THE TRANSLATIONAL MACHINERY

We showed previously that the sequence and (or) structural elements im portant for specifying the many distinctive properties of Escherichia coli initiator tRNA are clustered in the acceptor stem and in the anti codon stem and loop. This paper briefly describes this and reviews the results of some recently published studies on the mutant initiator tR NAs generated during this work. First, we have studied the effect of o verproduction of methionyl-tRNA transformylase (MTF) and initiation fa ctors IF2 and IF3 on activity of mutant initiator tRNAs that are defec tive at specific steps in the initiation pathway. Overproduction of MT F rescued specifically the activity of mutant tRNAs defective in formy lation but not mutants defective in binding to the P site. Overproduct ion of IF2 increased the activity of all mutant tRNAs having the CUA a nticodon but not of mutant tRNA having the GAC anticodon. Overproducti on of IF3 had no effect on the activity of any of the mutant tRNAs tes ted. Second, for functional studies of mutant initiator tRNA in vivo, we used a CAU-->CUA anticodon sequence mutant that can initiate protei n synthesis from UAG instead of AUG. In contrast with the wild-type in itiator tRNA, the mutant initiator tRNA has a 2-methylthio-N-6-isopent enyl adenosine (ms(2)i(6)A) base modification next to the anticodon. I nterestingly, this base modification is now important for activity of the mutant tRNA in initiation. In a miaA strain of E. coli deficient i n biosynthesis of ms(2)i(6)A, the mutant initiator tRNA is much less a ctive in initiation. The defect is specifically in binding to the ribo somal P site.