Mischarging mutants of Su⁺2 glutamine tRNA in E. coli.

I. Mutations near the anticodon cause mischarging

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In order to select the mischarging mutants of Su⁺2 glutamine tRNA, auxotrophic amber mutants of E. coli K12 which cannot be suppressed particularly by Su⁺2 were screened. By utilizing these mutants, cys_am235 and met_am3, several tens of mischarging mutants of Su⁺2 were isolated, as those conferring altered suppression patterns for a set of tester amber mutants of bacteria and phages. Nucleotide sequence analysis revealed that the mutation sites were found to be exclusively at Ψ37 residue located at the 3′-end of anticodon loop, changing it to either A37 or C37. These mutants were obtained as those suppressing cys_am235, and not met_am3. From these, secondary mutants were selected. In these mutants suppression patterns were further altered by the additional base substitutions, capable of suppressing met_am3. Such mutants were obtained exclusively from A37 and not from C37 mutant tRNA. Additional mutations to A37 were found to be either A29 or C38, which are located at the lowermost two base pairs in anticodon stem. The mischarging sites in Su⁺2 glutamine tRNA locate in the newly detected region of tRNA, differing from the previous case of Su⁺3 tyrosine or Su⁺7 tryptophan tRNAs. Implication of this finding is discussed on L-shaped tRNA molecule in relation to aminoacyl-tRNA synthetase recognition. Suppression patterns given by the double-mutants, A37A29 and A37C38, were consistent with the observation that the mutant tRNAs interact with tryptophanyl-tRNA synthetase.