Novel Escherichia coli RF1 mutants with decreased translation termination activity and increased sensitivity to the cytotoxic effect of the bacterial toxins Kid and RelE.

Molecular Microbiology
Elizabeth Diago-NavarroMarc Lemonnier

Abstract

Novel mutations in prfA, the gene for the polypeptide release factor RF1 of Escherichia coli, were isolated using a positive genetic screen based on the parD (kis, kid) toxin-antitoxin system. This original approach allowed the direct selection of mutants with altered translational termination efficiency at UAG codons. The isolated prfA mutants displayed a approximately 10-fold decrease in UAG termination efficiency with no significant changes in RF1 stability in vivo. All three mutations, G121S, G301S and R303H, were situated close to the nonsense codon recognition site in RF1:ribosome complexes. The prfA mutants displayed increased sensitivity to the RelE toxin encoded by the relBE system of E. coli, thus providing in vivo support for the functional interaction between RF1 and RelE. The prfA mutants also showed increased sensitivity to the Kid toxin. Since this toxin can cleave RNA in a ribosome-independent manner, this result was not anticipated and provided first evidence for the involvement of RF1 in the pathway of Kid toxicity. The sensitivity of the prfA mutants to RelE and Kid was restored to normal levels upon overproduction of the wild-type RF1 protein. We discuss these results and their utility for the design of nove...Continue Reading

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Citations

May 18, 2011·Antimicrobial Agents and Chemotherapy·Fernando BaqueroFernando de la Cruz
Aug 22, 2009·The FEBS Journal·Elizabeth Diago-NavarroRamón Díaz-Orejas
Aug 9, 2011·Critical Reviews in Biochemistry and Molecular Biology·Finbarr Hayes, Laurence Van Melderen
Sep 21, 2017·Scientific Reports·Laura Molina-García, Rafael Giraldo

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Methods Mentioned

BETA
electrophoresis
PCR

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