DNA-DEPENDENT RENATURATION OF AN INSOLUBLE DNA-BINDING PROTEIN - IDENTIFICATION OF THE RHAS BINDING-SITE AT RHABAD

Previous work has indicated that the RhaS protein directly activates t he L-rhamnose catabolic operon, rhaBAD, and that the likely RhaS bindi ng site lies downstream of position -84 relative to the rhaBBD transcr iption start point. Biochemical analysis of RhaS binding to this DNA s ite had not been possible due to the extreme insolubility of overprodu ced RhaS protein. Here we have been able to analyze directly the DNA b inding properties of RhaS by developing a method to refold insoluble R haS protein into a form with specific DNA binding activity. We found t hat active RhaS protein could be recovered only if the renaturation re action was performed in the presence of DNA. We also found that the re covery of DNA-binding activity from the related AraC protein, after de naturation in urea, was dependent upon added DNA. To test the specific ity of the recovered RhaS DNA binding activity, and to define the bind ing site for comparison with other AraC family binding sites, ive then investigated the details of the RhaS binding site. Using refolded Rha S protein in a DNase footprinting assay, we found that RhaS protects a region of the rhaBAD promoter from position -83 to -28. Analysis of t he effects of single base mutations in the rhaBAD promoter region indi cates that RhaS binds to an inverted repeat of two 17 bp half-sites se parated by 16 bp, located between -81 and -32 relative to the rhaBAD t ranscription start site.