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Free Content An altered-specificity DNA-binding mutant of Escherichia coli70 facilitates the analysis of 70 function in vivo

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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.
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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

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