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J. Biol. Chem., Vol. 281, Issue 51, 39407-39412, December 22, 2006

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LexA Represses CTX Transcription by Blocking Access of the C-terminal Domain of RNA Polymerase to Promoter DNA^*

Mariam Quinones, Harvey H. Kimsey, Wilma Ross, Richard L. Gourse, and Matthew K. Waldor¹

From the Department of Molecular Microbiology, Tufts University School of Medicine and the Howard Hughes Medical Institute, Boston, Massachusetts 02111 and Department of Bacteriology, University of Wisconsin, Madison, Wisconsin 53706

CTX is a Vibrio cholerae-specific temperate filamentous phage that encodes cholera toxin. CTX lysogens can be induced with DNA damage-inducing agents such as UV light, leading to the release of CTX virions and the rapid dissemination of cholera toxin genes to new V. cholerae hosts. This environmental regulation is directly mediated by LexA, the host-encoded global SOS transcription factor. LexA and a phage-encoded repressor, RstR, both repress transcription from P_rstA, the primary CTX promoter. Because the LexA binding site is located upstream of the core P_rstA promoter and overlaps with A-tract sequences, we speculated that LexA represses P_rstA by occluding a promoter UP element, a binding site for the C-terminal domain of the subunit of RNA polymerase (RNAP) (CTD). Using in vitro transcription assays, we have shown that the LexA binding site stimulates maximal rstA transcription in the absence of any added factors. The CTD of RNAP is required for this stimulation, demonstrating that the LexA site contains, or overlaps with, a promoter UP element. LexA represses rstA transcription by normal RNAP but fails to repress rstA transcription catalyzed by RNAP lacking the CTD. DNase I footprint analysis mapped the CTD binding site to the upstream promoter region that includes the LexA binding site. The addition of free subunits blocked the binding of LexA to rstA promoter DNA, indicating that LexA and the CTD directly compete for binding to their respective sites. To our knowledge, this is the first report of a repressor blocking transcription initiation by occluding a promoter UP element.

Received for publication, October 16, 2006

^* 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: Dept. of Molecular Microbiology, Tufts University School of Medicine, Boston, MA 02111. Tel.: 617-636-2730; Fax: 617-636-2723; E-mail: matthew.waldor{at}tufts.edu.

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