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J. Biol. Chem., Vol. 279, Issue 17, 17397-17403, April 23, 2004

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The Bacillus subtilis Response Regulator Spo0A Stimulates ^A-Dependent Transcription Prior to the Major Energetic Barrier^*

Steve D. Seredick and George B. Spiegelman

From the Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada

At the spoIIG promoter phosphorylated Spo0A (Spo0AP) binds 0A boxes overlapping the -35 element, interacting with RNA polymerase to facilitate open complex formation. We have compared in vitro transcription from a series of heteroduplex templates containing denatured regions within the promoters. Transcription from heteroduplex templates with 12, 8, or 6 base pairs denatured was independent of Spo0AP, but heteroduplexes with 4 or 2 base pairs denatured required Spo0AP for maximal levels of transcription. Investigation of the thermal dependence of transcription suggested that strand separation was the primary thermodynamic barrier to transcription initiation but indicated that Spo0AP does not reduce this energetic barrier. Kinetic assays revealed that Spo0AP stimulated both the rate of formation of initiated complexes as well as increasing the number of complexes capable of initiating transcription. These results imply that Spo0AP stimulates transcription at least in part by stabilizing the RNA polymerase-spoIIG complex until contacts between RNA polymerase and the -10 element induce strand separation.

Received for publication, October 10, 2003 , and in revised form, February 13, 2004.

^* This work was supported by grants from the Natural Science and Engineering Research Council of Canada and from the Canadian Institutes for Health Research (to G. B. 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 should be addressed: Dept. of Microbiology and Immunology, University of British Columbia, 6174 University Blvd., Vancouver, BC V6T 1Z3, Canada. Tel.: 604-822-2036; Fax: 604-822-6041; E-mail: spie{at}interchange.ubc.ca.

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