Novel Escherichia coli RF1 mutants with decreased translation termination activity and increased sensitivity to the cytotoxic effect of the bacterial toxins Kid and RelE.
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 novel antibacterial strategies in the light of the recently reported structure of ribosome-bound RF1.
Diago-Navarro
Elizabeth
E
Department of Molecular Microbiology, Centro de Investigaciones Biológicas CSIC, Ramiro de Maeztu 9, E-28040 Madrid, Spain.
Mora
Liliana
L
Buckingham
Richard H
RH
Díaz-Orejas
Ramón
R
Lemonnier
Marc
M
D001427:Bacterial Toxins / Q000378:metabolism*; D018388:Codon, Terminator; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D005816:Genetic Complementation Test; D016296:Mutagenesis; D010443:Peptide Chain Termination, Translational; D010454:Peptide Termination Factors / Q000235:genetics / Q000378:metabolism*
D001427:Bacterial Toxins; D018388:Codon, Terminator; D029968:Escherichia coli Proteins; D010454:Peptide Termination Factors; C465566:RelE protein, E coli; C473072:kid toxin protein, E coli plasmid R1; C489005:prfA protein, E coli