An altered-specificity DNA-binding mutant of Escherichia coli⁷⁰ facilitates the analysis of ⁷⁰ function in vivo

Summary

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