An altered-specificity DNA-binding mutant of Escherichia coli70 facilitates the analysis of 70 function in vivo
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.
Document Type: Research Article
Affiliations: 1: Department of Microbiology and Molecular Genetics, Harvard Medical School, 200 Longwood Ave., Boston, MA 02115, USA. 2: The Rockefeller University, 1230 York Ave., New York, NY 10021, USA.
Publication date: June 1, 2005