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J. Biol. Chem., Vol. 278, Issue 32, 29728-29743, August 8, 2003

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Mapping ⁵⁴-RNA Polymerase Interactions at the –24 Consensus Promoter Element^*

Patricia C. Burrows, Konstantin Severinov , Akira Ishihama , Martin Buck ¶ and Siva R. Wigneshweraraj ||

From the Department of Biological Sciences, Sir Alexander Fleming Building, Imperial College London, South Kensington Campus, London SW7 2AZ, United Kingdom, Waksman Institute and Department of Genetics, Rutgers, The State University, Piscataway, New Jersey 08904, and Nippon Institute for Biological Science, Shin-machi 9-2221, Ome, Tokyo 198-0024, Japan

The ⁵⁴ promoter specificity factor is distinct from ⁷⁰-type factors. The ⁵⁴-RNA polymerase binds to promoters with conserved sequence elements at –24 and –12 and utilizes specialized enhancer-binding activators to convert, through an ATP-dependent process, closed promoter complexes to open promoter complexes. The interface between ⁵⁴-RNA polymerase and promoter DNA is poorly characterized, contrasting with ⁷⁰. Here, ⁵⁴ was modified with strategically positioned cleavage reagents to provide physical evidence that the highly conserved RpoN box motif of ⁵⁴ is close to and may therefore interact with the consensus –24 promoter element. We show that the spatial relationship between the ⁵⁴-RNA polymerase and the –24 promoter element remains unchanged during closed to open complex conversion and transcription initiation but changes during the early elongation phase. In contrast, the spatial relationship between ⁵⁴-RNA polymerase and the consensus –12 promoter element changes upon conversion of the closed promoter complex to an open one. We provide evidence that some –12 promoter region-⁵⁴ interactions are dependent upon either the core RNA polymerase or a fork junction DNA structure at the –12-position, indicating that DNA fork junctions can substitute for core RNAP. We also show the -subunit flap domain contributes to different sets of -promoter DNA interactions at ⁵⁴- and ⁷⁰-dependent promoters.

Received for publication, April 7, 2003 , and in revised form, May 12, 2003.

^* This work was supported by a Biotechnology and Biological Sciences Research Council project grant (to M. B.) and National Institutes of Health Grant RO1 GM64530 (to K. S.). 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.

^¶ To whom correspondence may be addressed. Tel.: 44-207-594-5366; Fax: 44-207-594-5419; E-mail: m.buck{at}imperial.ac.uk.

^|| To whom correspondence may be addressed. Tel.: 44-207-594-5366; Fax: 44-207-594-5419; E-mail: s.r.wig{at}imperial.ac.uk.

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