title: Acquisition of a stable mutation in metY allows efficient initiation from an amber codon in Escherichia coli.
abstract: 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(1)(fMet), and a distantly located metY gene encodes a variant, tRNA(2)(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 tRNA(2)(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 tRNA(2)(fMet) (U35A36) with additional changes in the acceptor stem (G72; G72G73) but not in the anticodon stem (U29C30A31/U35A36/psi39G40A41). The usefulness of this strain, and a possible role for multiple tRNA(1)(fMet) genes in E. coli in safeguarding their intactness, are discussed.
authors.affiliation: Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560012, India.
authors.forename: Gautam
authors.initials: G
authors.lastname: Das
authors.affiliation: Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560012, India.
authors.forename: T K
authors.initials: TK
authors.lastname: Dineshkumar
authors.affiliation: Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560012, India.
authors.forename: Swapna
authors.initials: S
authors.lastname: Thanedar
authors.affiliation: Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560012, India.
authors.forename: Umesh
authors.initials: U
authors.lastname: Varshney
mesh_terms: D000926:Anticodon / Q000235:genetics*; D001483:Base Sequence; D018388:Codon, Terminator; D004926:Escherichia coli / Q000235:genetics*; D018628:Gene Dosage; D008969:Molecular Sequence Data; D010442:Peptide Chain Initiation, Translational / Q000235:genetics*; D010957:Plasmids; D017354:Point Mutation; D012329:RNA, Bacterial / Q000235:genetics / Q000502:physiology; D012358:RNA, Transfer, Met / Q000235:genetics* / Q000502:physiology; D011938:Rec A Recombinases
chemical_list: D000926:Anticodon; D018388:Codon, Terminator; D012329:RNA, Bacterial; D012358:RNA, Transfer, Met; D011938:Rec A Recombinases