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J. Bacteriol. doi:10.1128/JB.00096-08
Copyright (c) 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Promoter Activation by Repositioning of RNA polymerase

Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, GA 30322

^* To whom correspondence should be addressed. Email: moran{at}microbio.emory.edu.

	Abstract

Spo0A, a classic two-component type response regulator in B. subtilis, binds to a specific DNA sequence found in many promoters to repress or activate transcription of over 100 genes. On the spoIIG promoter, one of the Spo0A binding sites, centered at -40, overlaps a consensus -35 element that may also interact with region 4 of the sigma A (^A) subunit of the RNA polymerase. Molecular modeling corroborated by genetic evidence led us to propose that binding of Spo0A to this site repositions ^A region 4 on the promoter. Therefore, we used a chemical nuclease, p-bromoacetamidobenzyl-EDTA-Fe, that was covalently tethered to a single cysteine in region 4 of ^A to map the position of ^A on the promoter. The results indicated that in the absence of Spo0A, ^A region 4 of the RNA polymerase was located near the –35 element sequence centered at -40. However, in the presence of Spo0A, ^A region 4 was displaced downstream from the -35 element by 4 base pairs. These, and other results, support the model in which binding of Spo0A to the spoIIG promoter stimulates promoter utilization by repositioning prebound RNA polymerase, and stabilizing the repositioned RNA polymerase-promoter complex at a new position that aligns ^A region 2 with the -10 region sequences of the promoter, thus facilitating open complex formation.