Mischarging mutants of Su+2 glutamine tRNA in E. coli. II. Amino acid specificities of the mutant tRNAs.
Among the mischarging mutants isolated from strains with Su+2 glutamine tRNA, two double-mutants, A37A29 and A37C38, have been suggested to insert tryptophan at the UAG amber mutation site as determined by the suppression patterns of a set of tester mutants of bacteria and phages (Yamao et al., 1988). In this paper, we screened temperature sensitive mutants of E. coli in which the mischarging suppression was abolished even at the permissive temperature. Four such mutants were obtained and they were identified as the mutants of a structural gene for tryptophanyl-tRNA synthetase (trpS). Authentic trpS mutations, such as trpS5 or trpS18, also restricted the mischarging suppression. These results strongly support the previous prediction that the mutant tRNAs of Su+2, A37A29 and A37C38, are capable of interacting with tryptophanyl-tRNA synthetase and being misaminoacylated with tryptophan in vivo. However, in an assay to determine the specificity of the mutant glutamin tRNAs, we detected predominantly glutamine, but not any other amino acid, being inserted at an amber codon in vivo to any significant degree. We conclude that the mutant tRNAs still accept mostly glutamine, but can accept tryptophan in an extent for mischarging suppression. Since the amber suppressors of Su+7 tryptophan tRNA and the mischarging mutants of Su+3 tyrosine tRNA are charged with glutamine, structural similarity among the tRNAs for glutamine, tryptophan and tyrosine is discussed.
Yamao
F
F
Department of Biophysics, Faculty of Science, Kyoto University, Japan.
Inokuchi
H
H
Normanly
J
J
Abelson
J
J
Ozeki
H
H
D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics*; D005973:Glutamine / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation; D012345:RNA, Transfer, Amino Acid-Specific / Q000235:genetics*; D012351:RNA, Transfer, Gln / Q000235:genetics*; D012364:RNA, Transfer, Trp / Q000235:genetics; D012365:RNA, Transfer, Tyr / Q000235:genetics; D013489:Suppression, Genetic; D014369:Tryptophan-tRNA Ligase / Q000235:genetics
D012345:RNA, Transfer, Amino Acid-Specific; D012351:RNA, Transfer, Gln; D012364:RNA, Transfer, Trp; D012365:RNA, Transfer, Tyr; D005973:Glutamine; D014369:Tryptophan-tRNA Ligase