In vitro transcriptional analysis of the cytochrome P-450cam hydroxylase operon

To characterize the promoters of cytochrome P-450cam hydroxylase operon (ca mDCAB) and the repressor gene (camR), in vitro run-off transcription assays were performed using RNA polymerase (RNAP) holoenzyme reconstituted with t he core enzyme and the sigma(70) protein of Pseudomonas aeruginosa. Both th e mRNAs of camDCAB and camR were accurately transcribed from the respective promoter by the reconstituted RNAP holoenzyme, Both the transcriptions wer e repressed by CamR protein and the repressions were suppressed by D-campho r, consistent with the regulation in P. putida, These results suggest that the RNA polymerase containing sigma(70) recognizes the promoter of camDCAB as well as that of camR.