Transfer RNA identity change in anticodon variants of E. coli tRNAPhe in Vivo

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