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

Abstract

Escherichia colistrains 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 inE. coliresulted 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. coliK strains possess four initiator tRNA genes: themetZ,metWandmetVgenes, located at a single locus, encode tRNA1fMet, and a distantly locatedmetYgene encodes a variant, tRNA2fMet. In this study, a stable strain ofE. coliK-12 that affords efficient initiation from an amber initiation codon was isolated. Genetic analysis revealed that themetYgene in this strain acquired mutations to encode tRNA2fMetwith a CUA anticodon (a U35A36 mutation). The acquisition of the mutations depended on the presence of a plasmid-borne copy of the mutantmetYandrecA+host background. The mutations were observed when the plasmid-borne gene encoded tRNA2fMet(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 tRNA1fMetgenes inE. coliin safeguarding their intactness, are discussed.