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 containinlog 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_1^f^M^e^t, and a distantly located metY gene encodes a variant, tRNA_2^f^M^e^t. 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_2^f^M^e^t with a CUA anticodon (a U35A36 mutation). The acquisition of the mutations depended n the presence of a plasmid-borne copy of the mutant metY and recA+ host background. The mutations were observed when the plasmid-borne gene encoded tRNA_2^f^M^e^t (U35A36) with additional changes in the acceptor stem (G72; G72G73) but not in the anticodon stem (U29C30A31/U35A36/ \psi 39G40A41). The usefulness of this strain, and a possible role for multiple tRNA_1^f^M^e^t genes in E. coli in safeguarding their intactness, are discussed