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

Department of Molecular Biosciences, University of Kansas, Lawrence, KS 66045, USA; Transcription Control Section, Gene Regulation and Chromosome Biology Laboratory, Center for Cancer Research, National Cancer Institute at Frederick, NIH, Bldg. 469, PO Box B, Frederick, MD 21702, USA; Bioinformatics Core Facility, University of Kansas, Lawrence, KS 66045, USA

^* To whom correspondence should be addressed. Email: sme{at}ku.edu.

	Abstract

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 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.