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Acquisition of a Stable Mutation in Mety Allows Efficient Initiation from an Amber Codon in Escherichia Coli

Das, Gautam and Dineshkumar, TK and Thanedar, Swapan and Varshney, Umesh (2005) Acquisition of a Stable Mutation in Mety Allows Efficient Initiation from an Amber Codon in Escherichia Coli. In: Microbiology, 151 . pp. 1741-1750.


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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.

Item Type: Journal Article
Publication: Microbiology
Publisher: Society for General Microbiology
Additional Information: The copyright for this article belongs to Society for General Microbiology.
Department/Centre: Division of Biological Sciences > Microbiology & Cell Biology
Date Deposited: 24 Aug 2005
Last Modified: 19 Sep 2010 04:19
URI: http://eprints.iisc.ac.in/id/eprint/3541

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