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

Das, Gautam ; Dineshkumar, T. K. ; Thanedar, Swapna ; Varshney, Umesh (2005) Acquisition of a stable mutation in metY allows efficient initiation from an amber codon in Escherichia coli Microbiology, 151 (6). pp. 1741-1750. ISSN 1350-0872

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Official URL: http://mic.sgmjournals.org/content/151/6/1741.abst...

Related URL: http://dx.doi.org/10.1099/mic.0.27915-0

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 tRNA1fMet, and a distantly located metY gene encodes a variant, tRNA2fMet. 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 tRNA2fMet 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 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 in E. coli in safeguarding their intactness, are discussed.

Item Type:Article
Source:Copyright of this article belongs to Society for General Microbiology.
Keywords:Amp, Ampicillin; Cm, Chloramphenicol; CAT, Chloramphenicol Acetyl Transferase; Kan, Kanamycin; Tc, Tetracycline
ID Code:56281
Deposited On:23 Aug 2011 11:56
Last Modified:23 Aug 2011 11:56

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