Fujita M, Aramaki H, Horiuchi T, Amemura A
Department of Biotechnology, Faculty of Engineering, Fukuyama University, Hiroshima, Japan.
J Bacteriol. 1993 Nov;175(21):6953-8. doi: 10.1128/jb.175.21.6953-6958.1993.
In Pseudomonas putida carrying the CAM plasmid, the operon (camDCAB) encoding enzymes involved in the degradation pathway of D-camphor is negatively regulated by the CamR protein, and camR is autorepressed. S1 nuclease mapping revealed that camDCAB and camR were divergently transcribed from overlapping promoters, the transcription start sites were separated by 11 bp, and transcriptions of the cam operon (camDCAB) and camR increased about 10- and 4-fold, respectively, immediately after addition of camphor. The transcriptions of camDCAB and camR were negatively regulated through the interaction of the CamR protein with the one operator located in the overlapping promoter region. In vitro transcription experiments were performed to characterize the regulation of cam genes. The camR promoter was initiated by P. putida RNA polymerase containing sigma 70, but transcription from the camDCAB promoter by sigma 70 holoenzyme was not observed. The purified CamR protein repressed in vitro transcription from the camR promoter. This repression was suppressed by camphor. The RNA polymerase binding region of the camR promoter was identified by using DNase I footprinting. In addition, footprinting studies revealed that the CamR protein and RNA polymerase coexisted on the promoter region in a joint nonproductive complex.
在携带CAM质粒的恶臭假单胞菌中，编码参与D-樟脑降解途径的酶的操纵子（camDCAB）受CamR蛋白负调控，且camR自身被抑制。S1核酸酶图谱分析表明，camDCAB和camR从重叠启动子反向转录，转录起始位点相隔11 bp，添加樟脑后，cam操纵子（camDCAB）和camR的转录分别立即增加约10倍和4倍。camDCAB和camR的转录通过CamR蛋白与位于重叠启动子区域的一个操纵子相互作用而受到负调控。进行了体外转录实验以表征cam基因的调控。camR启动子由含σ70的恶臭假单胞菌RNA聚合酶起始，但未观察到σ70全酶从camDCAB启动子进行转录。纯化的CamR蛋白抑制camR启动子的体外转录。这种抑制被樟脑抑制。通过DNase I足迹法鉴定了camR启动子的RNA聚合酶结合区域。此外，足迹研究表明，CamR蛋白和RNA聚合酶以联合非生产性复合物的形式共存于启动子区域。
