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J. Biol. Chem., Vol. 280, Issue 46, 38219-38227, November 18, 2005

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Escherichia coli RNA Polymerase Contacts outside the –10 Promoter Element Are Not Essential for Promoter Melting^*

From the Edward A. Doisy Department of Biochemistry and Molecular Biology, St. Louis University Medical School, St. Louis, Missouri 63104

We examined the relative affinity of model promoter constructs for binding Escherichia coli RNA polymerase (RNAP) holoenzyme. Model promoter constructs were designed to mimic DNA structures characteristic for different steps of transcription initiation. DNA duplexes in which a chemical cross-link was introduced just downstream from –10 hexamer to prevent DNA melting upon RNAP binding were used to mimic RNAP-promoter contacts in a closed complex. Fork junction DNA molecules with double-stranded/single-stranded junction between –11 and –10 position were used to study interactions of RNA polymerase with DNA in open complex. The –35 and –10 promoter regions were found to be equally important for the initial RNAP binding. The recognition of –35 promoter region was independent of structural context of the model promoter fragment. In contrast, free energy of RNAP binding to –10 hexamer was highly dependent on DNA structure. The relative importance of –10 region for sequence-specific interaction with the polymerase was the lowest for constructs mimicking closed complex and the highest for the constructs mimicking open complex. The relative importance of region –10 was also dependent on the presence of –35 consensus element indicating a communication between different DNA binding determinants of polymerase during open complex formation. Short double-stranded promoter fragments comprising only –35 and –10 or only –10 consensus elements underwent melting in a complex with polymerase indicating that the core of promoter melting activity of the polymerase is localized to a very small subset of all promoter-polymerase contacts.

Received for publication, July 21, 2005 , and in revised form, September 12, 2005.

^* This work was supported by National Institutes of Health Grant GM 50514. 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 On leave from the Dept. of Organic Chemistry, Biochemistry, and Biotechnology, Wroclaw University of Technology, 50370 Wroclaw, Poland. Present address: Dept. of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO 63110.

² To whom correspondence should be addressed: Edward A. Doisy Dept. of Biochemistry and Molecular Biology, St. Louis University School of Medicine, 1402 S. Grand Blvd., St. Louis, MO 63104. Tel.: 314-977-9238; Fax: 314-977-9205; E-mail: heydukt{at}slu.edu.

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