Webster C, Gaston K, Busby S
Department of Biochemistry, University of Birmingham, U.K.
Gene. 1988 Sep 7;68(2):297-305. doi: 10.1016/0378-1119(88)90032-7.
Expression of the melR gene is required for melibiose-dependent stimulation of transcription initiation at the promoter of the melAB operon. Using the S1 nuclease method we have located the melR transcription start point. Transcription from the melR promoter is dependent on cAMP-CRP: specific nucleotide sequences downstream of bp -59 with respect to the melR transcription start are sufficient for full promoter activity. Nucleotide sequence homologies suggest that the cAMP-CRP binding site is located from bp -52 to -31, in exactly the same position as at the galP1 promoter. Using DNase I footprinting we show that cAMP-CRP and RNA polymerase together bind tightly to the melR promoter sequence, creating a strong footprint from bp -70 to +20. Alone, cAMP-CRP binding is hardly detectable, whereas RNA polymerase alone creates a weak footprint centred around the -10 hexamer sequence. When the melR gene is expressed from a cAMP-CRP-independent promoter, melibiose-dependent transcription from the melAB promoter becomes independent of cAMP-CRP, showing that the melR promoter is the primary site of control by cAMP-CRP in the mel regulon.
melR基因的表达是蜜二糖依赖性刺激melAB操纵子启动子转录起始所必需的。我们使用S1核酸酶法确定了melR转录起始点。melR启动子的转录依赖于cAMP-CRP：相对于melR转录起始点，-59 bp下游的特定核苷酸序列足以实现完整的启动子活性。核苷酸序列同源性表明，cAMP-CRP结合位点位于-52至-31 bp，与galP1启动子的位置完全相同。使用DNase I足迹法，我们发现cAMP-CRP和RNA聚合酶一起紧密结合到melR启动子序列上，从-70至+20 bp产生强烈的足迹。单独时，几乎检测不到cAMP-CRP的结合，而单独的RNA聚合酶会在-10六聚体序列周围产生较弱的足迹。当melR基因由不依赖cAMP-CRP的启动子表达时，melAB启动子的蜜二糖依赖性转录变得不依赖于cAMP-CRP，这表明melR启动子是mel操纵子中cAMP-CRP控制的主要位点。
