Transcription activation by the DNA-binding domain of the AraC family protein RhaS in the absence of its effector-binding domain.
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 DeltarhaS 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.
Wickstrum
Jason R
JR
Department of Molecular Biosciences, 1200 Sunnyside Ave., University of Kansas, Lawrence, KS 66045, USA.
Skredenske
Jeff M
JM
Kolin
Ana
A
Jin
Ding J
DJ
Fang
Jianwen
J
Egan
Susan M
SM
D051777:AraC Transcription Factor / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D015153:Blotting, Western; D002373:Cyclic AMP Receptor Protein / Q000235:genetics / Q000378:metabolism; D018983:DNA Footprinting / Q000379:methods; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D011485:Protein Binding; D018085:Regulon / Q000235:genetics; D012210:Rhamnose / Q000378:metabolism; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism; D015533:Transcriptional Activation
D051777:AraC Transcription Factor; C497371:AraC protein, E coli; D002373:Cyclic AMP Receptor Protein; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C062863:RhaR protein, E coli; C084095:RhaS protein, E coli; D015534:Trans-Activators; D012210:Rhamnose