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Journal of Bacteriology, April 2000, p. 1995-2000, Vol. 182, No. 7
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Cooperative Action of the Catabolite Activator Protein and AraC In Vitro at the araFGH Promoter

Casonya M. Johnsondagger and Robert F. Schleif*

Department of Biology, Johns Hopkins University, Baltimore, Maryland 21218

Received 20 September 1999/Accepted 12 January 2000

Full activation of transcription of the araFGH promoter, pFGH, requires both the catabolite activator protein (CAP) and AraC protein. At pFGH, the binding site for CAP is centered at position -41.5, an essential binding site for AraC is centered at position -79.5, and a second, nonessential binding site is centered at position -154.5. In this work, we used the minimal promoter region required for in vivo activation of pFGH to examine the roles of CAP and AraC in stimulating formation of open complexes at pFGH. Migration retardation assays of open complexes showed that RNA polymerase binds exceptionally tightly to the AraC-CAP-pFGH complex and that the order of addition of proteins to the initiating complex is important. Similar assays with RNA polymerase containing truncated alpha subunits suggest that AraC interacts with the C-terminal domain of the alpha subunit. Finally, AraC protein also acts to prevent the improper binding of RNA polymerase at a pseudo promoter near the true pFGH promoter.

* Corresponding author. Mailing address: Department of Biology, Johns Hopkins University, 3400 N. Charles St., Baltimore, MD 21218. Phone: (410) 516-5206. Fax: (410) 516-5213. E-mail: bio_zrfs{at}jhuvms.hcf.jhu.edu.

dagger Present address: Biology Department, Morgan State University, Baltimore, MD 21251.

Journal of Bacteriology, April 2000, p. 1995-2000, Vol. 182, No. 7
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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