DNA-BINDING CHARACTERISTICS OF CRTJ - A REDOX-RESPONDING REPRESSOR OFBACTERIOCHLOROPHYLL, CAROTENOID, AND LIGHT HARVESTING-II GENE-EXPRESSION IN RHODOBACTER-CAPSULATUS

Previous genetic analysis indicated that the photosynthesis gene clust er from Rhodobacter capsulatus coded for the transcription factor, Crt J, that is responsible for aerobic repression of bacteriochlorophyll, carotenoid, and light harvesting-II gene expression. In this study, we have heterologously overexpressed and purified CrtJ to homogeneity an d shown by gel mobility shift assays that CrtJ is biologically active. DNase I footprint analysis confirms molecular genetic studies by show ing that CrtJ binds to conserved palindromic sequences that overlap th e -10 and -35 promoter regions of the bchC operon. Graphs of the perce ntage of DNA bound versus protein concentration show sigmoidal curves, which is highly indicative of cooperative binding of CrtJ to the two palindromic sites. A binding constant for interaction of CrtJ with the palindrome that spans the -10 region was calculated to be 4.8 x 10(-9 ) M, whereas affinity for the palindrome that spans the -35 region was found to be 2.9 x 10(-9) hi. Binding of CrtJ to the bchC promoter reg ion was also found to be redox-sensitive, with CrtJ exhibiting a 4.5-f old higher binding affinity under oxidizing versus reducing conditions .