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J. Biol. Chem., Vol. 281, Issue 15, 10049-10055, April 14, 2006

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MarA-mediated Transcriptional Repression of the rob Promoter^*

Thamarai Schneiders and Stuart B. Levy¹

From the Center for Adaptation Genetics and Drug Resistance and the Departments of Molecular Biology and Microbiology and of Medicine, Tufts University School of Medicine, Boston, Massachusetts 02111

The Escherichia coli transcriptional regulator MarA affects functions that include antibiotic resistance, persistence, and survival. MarA functions as an activator or repressor of transcription utilizing similar degenerate DNA sequences (marboxes) with three different binding site configurations with respect to the RNA polymerase-binding sites. We demonstrate that MarA down-regulates rob transcripts both in vivo and in vitro via a MarA-binding site within the rob promoter that is positioned between the –10 and –35 hexamers. As for the hdeA and purA promoters, which are repressed by MarA, the rob marbox is also in the "backward" orientation. Protein-DNA interactions show that SoxS and Rob, like MarA, bind the same marbox in the rob promoter. Electrophoretic mobility shift analyses with a MarA-specific antibody demonstrate that MarA and RNA polymerase form a ternary complex with the rob promoter DNA. Transcription experiments in vitro and potassium permanganate footprinting analysis show that MarA affects the RNA polymerase-mediated closed to open complex formation at the rob promoter.

Received for publication, November 9, 2005 , and in revised form, February 8, 2006.

^* This work was supported by United States Public Health Service Grant AI56021 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Center for Adaptation Genetics and Drug Resistance, Tufts University School of Medicine, 136 Harrison Ave., Boston, MA 02111. Tel.: 617-636-6764; Fax: 617-636-0458; E-mail: stuart.levy{at}tufts.edu.

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