An altered-specificity DNA-binding mutant of Escherichia coli σ70 facilitates the analysis of σ70 function in vivo

The σ subunit of bacterial RNA polymerase is strictly required for promoter recognition. The primary (housekeeping) σ factor of Escherichia coli, σ(70), is responsible for most of the gene expression in exponentially growing cells. The fact that σ(70) is an essential protein has complicated efforts to genetically dissect the functions of σ(70). To facilitate the analysis of σ(70) function in vivo, we isolated an altered-specificity DNA-binding mutant of σ(70), σ(70) R584A, which preferentially recognizes a mutant promoter that is not efficiently recognized by wild-type σ(70). Exploiting this σ(70) mutant as a genetic tool, we establish an in vivo assay for the inhibitory effect of the bacteriophage T4-encoded anti-σ factor AsiA on σ(70)-dependent transcription. Our results demonstrate the utility of this altered-specificity system for genetically dissecting σ(70) and its interactions with transcription regulators.