Mustaev A, Zaychikov E, Severinov K, Kashlev M, Polyakov A, Nikiforov V, Goldfarb A
Public Health Research Institute, New York, NY 10016.
Proc Natl Acad Sci U S A. 1994 Dec 6;91(25):12036-40. doi: 10.1073/pnas.91.25.12036.
Spatial organization of the binding sites for the priming substrate, the template DNA, and the transcription inhibitor rifampicin (Rif) in Escherichia coli RNA polymerase (EC 2.7.7.6) was probed with chimeric compounds in which Rif is covalently attached to a ribonucleotide. The compounds bind to RNA polymerase in bifunctional manner and serve as substrates for RNA chain extension, yielding chains up to 8 nucleotides in length, with Rif linked to their 5' termini. These products act as potent inhibitors of normal transcription. Using the linker between the two ligands as ruler, we determined the distance between the sites for Rif and the priming nucleotide to be approximately 15 A. A reactive side group placed in the linker next to Rif crosslinks to the template strand of DNA at the -2 or -3 position of the promoter. Thus, bound Rif is juxtaposed to DNA immediately upstream of the start site, suggesting that Rif plugs the channel leading RNA out of the active center.
利用嵌合化合物对大肠杆菌RNA聚合酶（EC 2.7.7.6）中引发底物、模板DNA和转录抑制剂利福平（Rif）的结合位点的空间组织进行了探测，在这些嵌合化合物中，利福平与核糖核苷酸共价连接。这些化合物以双功能方式与RNA聚合酶结合，并作为RNA链延伸的底物，产生长度达8个核苷酸的链，利福平连接在其5'末端。这些产物是正常转录的有效抑制剂。以两个配体之间的连接体为尺子，我们确定利福平和引发核苷酸的位点之间的距离约为15埃。位于利福平旁边连接体中的一个反应性侧基在启动子的-2或-3位置与DNA的模板链交联。因此，结合的利福平紧邻起始位点上游的DNA，这表明利福平堵塞了将RNA引出活性中心的通道。
