Pfeffer S R, Stahl S J, Chamberlin M J
J Biol Chem. 1977 Aug 10;252(15):5403-7.
Escherichia coli RNA polymerase holoenzyme bound to promoter sites on T7 DNA is attacked and inactivated by the polyanion heparin. The highly stable RNA polymerase-T7 DNA complex formed at the major T7 A1 promoter can be completely inactivated by treatment with heparin, as shown by monitoring the loss of activity of such complexes, and by gel electrophoresis of the RNA products transcribed. The rate of this inactivation is much faster than the rate of dissociation of RNA polymerase from promoter complexes, and thus represents a direct attack of heparin on the polymerase molecule bound at promoter A1. Experiments employing the nitrocellulose filter binding technique suggest that heparin inactivates E. coli RNA polymerase when bound to T7 DNA by directly displacing the enzyme from the DNA. RNA polymerase bound at a minor T7 promoter (promoter C) is much less sensitive to heparin attack than enzyme bound at promoter A1. Thus, the rate of inactivation of RNA polymerase-T7 DNA complexes by heparin is dependent upon the structure of the promoter involved even though the inhibitor binds to a site on the enzyme molecule.
与T7 DNA上启动子位点结合的大肠杆菌RNA聚合酶全酶会受到多聚阴离子肝素的攻击并失活。如通过监测此类复合物活性的丧失以及对转录的RNA产物进行凝胶电泳所示，在主要的T7 A1启动子处形成的高度稳定的RNA聚合酶-T7 DNA复合物可被肝素处理完全失活。这种失活的速率比RNA聚合酶从启动子复合物上解离的速率快得多，因此代表了肝素对结合在启动子A1处的聚合酶分子的直接攻击。采用硝酸纤维素滤膜结合技术的实验表明，肝素与T7 DNA结合时会通过直接将酶从DNA上置换下来而使大肠杆菌RNA聚合酶失活。结合在次要T7启动子（启动子C）上的RNA聚合酶比结合在启动子A1上的酶对肝素攻击的敏感性要低得多。因此，尽管抑制剂与酶分子上的一个位点结合，但肝素使RNA聚合酶-T7 DNA复合物失活的速率取决于所涉及启动子的结构。
