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Journal of Bacteriology, July 2007, p. 4984-4993, Vol. 189, No. 14
0021-9193/07/$08.00+0     doi:10.1128/JB.00530-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Transcription Activation by the DNA-Binding Domain of the AraC Family Protein RhaS in the Absence of Its Effector-Binding Domain

Jason R. Wickstrum,^1, Jeff M. Skredenske,¹ Ana Kolin,^1, Ding J. Jin,² Jianwen Fang,³ and Susan M. Egan^1*

Department of Molecular Biosciences, University of Kansas, Lawrence, Kansas 66045,¹ Transcription Control Section, Gene Regulation and Chromosome Biology Laboratory, Center for Cancer Research, National Cancer Institute at Frederick, NIH, Bldg. 469, P.O. Box B, Frederick, Maryland 21702,² Bioinformatics Core Facility, University of Kansas, Lawrence, Kansas 66045³

Received 6 April 2007/ Accepted 9 May 2007

The Escherichia coli L-rhamnose-responsive transcription activators RhaS and RhaR both consist of two domains, a C-terminal DNA-binding domain and an N-terminal dimerization domain. Both function as dimers and only activate transcription in the presence of L-rhamnose. Here, we examined the ability of the DNA-binding domains of RhaS (RhaS-CTD) and RhaR (RhaR-CTD) to bind to DNA and activate transcription. RhaS-CTD and RhaR-CTD were both shown by DNase I footprinting to be capable of binding specifically to the appropriate DNA sites. In vivo as well as in vitro transcription assays showed that RhaS-CTD could activate transcription to high levels, whereas RhaR-CTD was capable of only very low levels of transcription activation. As expected, RhaS-CTD did not require the presence of L-rhamnose to activate transcription. The upstream half-site at rhaBAD and the downstream half-site at rhaT were found to be the strongest of the known RhaS half-sites, and a new putative RhaS half-site with comparable strength to known sites was identified. Given that cyclic AMP receptor protein (CRP), the second activator required for full rhaBAD expression, cannot activate rhaBAD expression in a rhaS strain, it was of interest to test whether CRP could activate transcription in combination with RhaS-CTD. We found that RhaS-CTD allowed significant activation by CRP, both in vivo and in vitro, although full-length RhaS allowed somewhat greater CRP activation. We conclude that RhaS-CTD contains all of the determinants necessary for transcription activation by RhaS.

Published ahead of print on 18 May 2007.

Present address: Department of Microbiology, Molecular Genetics and Immunology, University of Kansas Medical Center, Kansas City, KS 66160.

Present address: Department of Microbiology and Immunology, 4301 Jones Bridge Road, Uniformed Services University of the Health Sciences, Bethesda, MD 20814.

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