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

Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560012, India

Correspondence
Umesh Varshney
varshney{at}mcbl.iisc.ernet.in

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 tRNA₁^fMet, and a distantly located metY gene encodes a variant, tRNA₂^fMet. 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 tRNA₂^fMet 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⁺ host background. The mutations were observed when the plasmid-borne gene encoded tRNA₂^fMet (U35A36) with additional changes in the acceptor stem (G72; G72G73) but not in the anticodon stem (U29C30A31/U35A36/39G40A41). The usefulness of this strain, and a possible role for multiple tRNA₁^fMet genes in E. coli in safeguarding their intactness, are discussed.