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Journal of Bacteriology, May 2008, p. 3110-3117, Vol. 190, No. 9
0021-9193/08/$08.00+0     doi:10.1128/JB.00096-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Promoter Activation by Repositioning of RNA Polymerase{triangledown}

Amrita Kumar and Charles P. Moran Jr.*

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

Received 18 January 2008/ Accepted 13 February 2008

Spo0A, a classical two-component-type response regulator in Bacillus subtilis, binds to a specific DNA sequence found in many promoters to repress or activate the transcription of over 100 genes. On the spoIIG promoter, one of the Spo0A binding sites, centered at position –40, overlaps a consensus –35 element that may also interact with region 4 of the sigma A ({sigma}A) subunit of RNA polymerase. Molecular modeling corroborated by genetic evidence led us to propose that the binding of Spo0A to this site repositions {sigma}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 {sigma}A to map the position of {sigma}A on the promoter. The results indicated that in the absence of Spo0A, {sigma}A region 4 of the RNA polymerase was located near the –35 element sequence centered at position –40. However, in the presence of Spo0A, {sigma}A region 4 was displaced downstream from the –35 element by 4 bp. These and other results support the model in which the 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 {sigma}A region 2 with the –10 region sequences of the promoter, thus facilitating open complex formation.

* Corresponding author. Mailing address: Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, GA 30322. Phone: (404) 727-5969. Fax: (404) 727-3659. E-mail: moran{at}microbio.emory.edu

{triangledown} Published ahead of print on 22 February 2008.

Journal of Bacteriology, May 2008, p. 3110-3117, Vol. 190, No. 9
0021-9193/08/$08.00+0     doi:10.1128/JB.00096-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



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