Sur R, Debnath D, Mukhopadhyay J, Parrack P
Department of Biochemistry, Bose Institute, P 1/12, C.I.T. Scheme VIIM, Calcutta, India.
Eur J Biochem. 2001 Apr;268(8):2344-50. doi: 10.1046/j.1432-1327.2001.02114.x.
RNA polymerase is known to bind and utilize the overlapping promoters P1 and P2 in Escherichia coli galactose operon. We have identified an additional specific site upstream of P2, where RNA polymerase binds in a heparin-resistant manner. Binding of polymerase to this site, termed P3, occurs simultaneous to its binding at P1/P2. We have located this P3 site by DNase I footprinting. A 63 base pair region centered around position - 100 with respect to galP1 is protected by polymerase. Interestingly, a Pribnow box TATAAT is present within this protected region (-103 to -108). We have shown that transcription occurs from P3 in vitro. Primer extension analysis provides direct evidence that P3 is transcribed in vivo. The start site of transcription has been mapped at -96 position relative to galP1. beta-galactosidase assays with different gal promoter constructs reveal that while P3 alone functions as a weak in vivo promoter, it has a synergistic effect on transcription from the gal operon, since deletion of P3 or specifically mutating its -10 region result in a substantial reduction in the gal promoter activity.
已知RNA聚合酶能结合并利用大肠杆菌半乳糖操纵子中重叠的启动子P1和P2。我们在P2上游鉴定出了一个额外的特定位点，RNA聚合酶以抗肝素的方式结合于此。聚合酶与这个被称为P3的位点的结合与其在P1/P2处的结合同时发生。我们通过DNA酶I足迹法确定了这个P3位点。相对于galP1，一个以-100位置为中心的63个碱基对的区域受到聚合酶的保护。有趣的是，在这个受保护区域（-103至-108）内存在一个Pribnow框TATAAT。我们已经证明在体外转录从P3开始。引物延伸分析提供了直接证据表明P3在体内被转录。转录起始位点已定位在相对于galP1的-96位置。用不同的半乳糖启动子构建体进行的β-半乳糖苷酶测定表明，虽然单独的P3作为一个弱的体内启动子起作用，但它对来自半乳糖操纵子的转录有协同作用，因为删除P3或特异性突变其-10区域会导致半乳糖启动子活性大幅降低。
