Acquisition of a stable mutation in metY allows efficient initiation from an amber codon in Escherichia coli

Escherichia coli strains harbouring elongator tRNAs that insert amino acids in response to a termination codon during elongation have been generated for various applications. Additionally, it was shown that expression of an initiator tRNA containing a CUA anticodon from a multicopy plasmid in E. coli resulted in initiation from an amber codon. Even though the initiation-based system remedies toxicity-related drawbacks, its usefulness has remained limited for want of a strain with a chromosomally encoded initiator tRNA 'suppressor'. E. coli K strains possess four initiator tRNA genes: the metZ, metW and metV genes, located at a single locus, encode tRNA<SUB>1</SUB><SUP>fMet</SUP>, and a distantly located metY gene encodes a variant, tRNA<SUB>2</SUB><SUP>fMet</SUP>. In this study, a stable strain of E. coli K-12 that affords efficient initiation from an amber initiation codon was isolated. Genetic analysis revealed that the metY gene in this strain acquired mutations to encode tRNA<SUB>2</SUB><SUP>fMet</SUP> with a CUA anticodon (a U35A36 mutation). The acquisition of the mutations depended on the presence of a plasmid-borne copy of the mutant metY and recA<SUP>+</SUP>host background. The mutations were observed when the plasmid-borne gene encoded tRNA<SUB>2</SUB><SUP>fMet</SUP> (U35A36) with additional changes in the acceptor stem (G72; G72G73) but not in the anticodon stem (U29C30A31/U35A36/ψ39G40A41). The usefulness of this strain, and a possible role for multiple tRNA<SUB>1</SUB><SUP>fMet</SUP>genes in E. coli in safeguarding their intactness, are discussed