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J. Biol. Chem., Vol. 278, Issue 34, 31701-31708, August 22, 2003

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RNA Polymerase Subunit Requirements for Activation by the Enhancer-binding Protein Rhodobacter capsulatus NtrC^*

Cynthia L. Richard, Animesh Tandon, Nathaniel R. Sloan and Robert G. Kranz 

From the Department of Biology, Washington University, St. Louis, Missouri 63130

Rhodobacter capsulatus NtrC is an enhancer-binding protein that activates transcription of the R. capsulatus ⁷⁰ RNA polymerase, but does not activate the Escherichia coli ⁷⁰-RNA polymerase at the nifA1 promoter. We utilized R. capsulatus:E. coli hybrid RNA polymerases assembled in vitro to investigate the subunits required for protein-protein interaction with RcNtrC at the nifA1mut1 promoter. Assembly of core Rc' or hybrid RNA polymerases containing the Rc' subunits absolutely require the inclusion of an subunit, with the Ec subunit only partially promoting RNA polymerase assembly. The RcEc' RNA polymerase is not activated by RcNtrC. Moreover, a mutant form of the Rc lacking the C-terminal domain, when assembled with the Rc' and ⁷⁰ subunits, is activated by RcNtrC. These results suggest that the R. capsulatus subunit is not important for RcNtrC interaction. All hybrid RNA polymerases that contained the Rc' were activated by RcNtrC, suggesting that the Rc' subunit plays an important role. It is proposed that RcNtrC recruits R. capsulatus ⁷⁰-RNA polymerase to the promoter through interaction with Rc'. RcNtrC interacts with RNA polymerase from a unique position, with dimers centered at –118 bp from the start site. Placing the RcNtrC tandem binding sites on the opposite face of the helix (–113 bp) completely abolished transcription activation. Moving the RcNtrC tandem binding sites 20 bp closer to or further from the promoter significantly reduced activation, again suggesting unique spatial constraints on how RcNtrC interacts with the R. capsulatus RNA polymerase.

Received for publication, April 28, 2003 , and in revised form, May 22, 2003.

^* This work was supported by United States Department of Agriculture NRI Grant 99-35305-8647 (to R. G. K.). 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. Tel.: 314-935-4278; Fax: 314-935-4432; E-mail: kranz{at}biology.wustl.edu.

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