Transfer RNA identity change in anticodon variants of E. coil tRNA(Phe) inVivo

The anticodon sequence is a major recognition element for most aminoacyl-tR NA synthetases, We investigated the in vivo effects of changing the anticod on on the aminoacylation specificity in the example of E. coil tRNA(Phe). C onstructing different anticodon mutants of E. coli tRNA(Phe) by site-direct ed mutagenesis, we isolated 22 anticodon mutant tRNA(Phe); the anticodons c orresponded to 16 amino acids and an opal stop codon, To examine whether th e mutant tRNAs had changed their amino acid acceptor specificity in vivo, w e tested the viability off. coil strains containing these tRNA(Phe) genes i n a medium which permitted tRNA induction. Fourteen mutant tRNA genes did n ot affect host viability, However, eight mutant tRNA genes were toxic to th e host and prevented growth, presumably because the anticodon mutants led t o translational errors. Many mutant tRNAs which did not affect host viabili ty were not aminoacylated in vivo. Three mutant tRNAs containing anticodon sequences corresponding to lysine (UUU), methionine (CAU) and threonine (UG U) were charged with the amino acid corresponding to their anticodon, but n ot with phenylalanine, These three tRNAs and tRNA(Phe) are located in the s ame cluster in a sequence similarity dendrogram of total E. coli tRNAs, The results support the idea that such tRNAs arising from in vivo evolution ar e derived by anticodon change from the same ancestor tRNA.