An altered-specificity DNA-binding mutant of Escherichia coli sigma70 facilitates the analysis of sigma70 function in vivo.
The sigma subunit of bacterial RNA polymerase is strictly required for promoter recognition. The primary (housekeeping) sigma factor of Escherichia coli, sigma(70), is responsible for most of the gene expression in exponentially growing cells. The fact that sigma(70) is an essential protein has complicated efforts to genetically dissect the functions of sigma(70). To facilitate the analysis of sigma(70) function in vivo, we isolated an altered-specificity DNA-binding mutant of sigma(70), sigma(70) R584A, which preferentially recognizes a mutant promoter that is not efficiently recognized by wild-type sigma(70). Exploiting this sigma(70) mutant as a genetic tool, we establish an in vivo assay for the inhibitory effect of the bacteriophage T4-encoded anti-sigma factor AsiA on sigma(70)-dependent transcription. Our results demonstrate the utility of this altered-specificity system for genetically dissecting sigma(70) and its interactions with transcription regulators.
Gregory
Brian D
BD
Department of Microbiology and Molecular Genetics, Harvard Medical School, 200 Longwood Ave., Boston, MA 02115, USA.
Nickels
Bryce E
BE
Darst
Seth A
SA
Hochschild
Ann
A
D019943:Amino Acid Substitution; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000235:genetics* / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D017930:Genes, Reporter; D008958:Models, Molecular; D009154:Mutation; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D012808:Sigma Factor / Q000235:genetics* / Q000378:metabolism*; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism; D001616:beta-Galactosidase / Q000032:analysis / Q000235:genetics
C078876:AsiA protein, Enterobacteria phage T4; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D012808:Sigma Factor; D014764:Viral Proteins; C081775:RNA polymerase sigma 70; D012321:DNA-Directed RNA Polymerases; D001616:beta-Galactosidase