DNA-dependent renaturation of an insoluble DNA binding protein. Identification of the RhaS binding site at rhaBAD.

摘要: Previous work has indicated that the RhaS protein directly activates L-rhamnose catabolic operon, rhaBAD, and likely binding site lies downstream of position -84 relative to rhaBAD transcription start point. Biochemical analysis this DNA had not been possible due extreme insolubility overproduced protein. Here we have able analyze properties by developing a method refold insoluble into form with specific activity. We found active could be recovered only if renaturation reaction was performed in presence DNA. also recovery DNA-binding activity from related AraC protein, after denaturation urea, dependent upon added To test specificity activity, define for comparison other family sites, then investigated details site. Using refolded DNase footprinting assay, protects region promoter -83 -28. Analysis effects single base mutations indicates binds an inverted repeat two 17 bp half-sites separated 16 bp, located between -81 -32