Interactions of transcription inhibitors with the Escherichia coli RNA polymerase-lacUV5 promoter open complex.
Interactions of transcription inhibitors with the Escherichia coli RNA polymerase-lacUV5 promoter open complex.
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DOI：
10.1021/bi00174a037
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发表时间：
1994-03
期刊：
影响因子：
2.9
通讯作者：
A. Mazumder;D. Perrin;D. McMillin;D. Sigman
中科院分区：
文献类型：
--
作者：
A. Mazumder;D. Perrin;D. McMillin;D. Sigman
用凝胶阻滞法、1，10-菲咯啉（OP）及其衍生物的亚铜配合物的化学核酸酶活性和稳态动力学研究了转录抑制剂与大肠杆菌RNA聚合酶和lacUV 5启动子组成的开放复合物的相互作用。凝胶阻滞显示2：1的2，9-二甲基-1，10-菲咯啉-亚铜配合物[（2，9-Me_2OP）_2Cu ~+]和利福平能稳定地与该开放配合物结合。（2，9-Me 2 OP）2Cu+通过干扰其氧化还原活性电子等排体的结合来阻断化学核酸酶的断裂。利福平不阻断3，4，7，8-四甲基-OP、4-苯基-OP和OP的亚铜络合物的断裂，但干扰5-苯基-OP的亚铜络合物的断裂。柔红霉素、二（脒基苯基）吲哚（DAPI）、放线菌素D、偏端霉素、9-氨基吖啶、光辉霉素和色霉素A3），它们以高亲和力结合游离DNA，不与开放复合物形成稳定的三元复合物。凝胶阻滞实验表明，它们促进酶与启动子的解离。相对于聚合酶结合，酶催化对抑制剂浓度的更大的敏感性表明这些配体与RNA聚合酶和启动子形成亚稳态的、无催化活性的三元复合物。
The interactions of transcription inhibitors with the open complex composed of Escherichia coli RNA polymerase and the lacUV5 promoter have been studied using gel retardation, the chemical nuclease activity of the cuprous complexes of 1,10-phenanthroline (OP) and its derivatives, and steady-state kinetics. Gel retardation shows that two inhibitors, the 2:1 2,9-dimethyl-1,10-phenanthroline-cuprous complex [(2,9-Me2OP)2Cu+] and rifampicin, bind stably to the open-complex. (2,9-Me2OP)2Cu+ blocks scission by the chemical nuclease by interfering with the binding of its redox-active isosteres. Rifampicin does not block scission by the cuprous complexes of 3,4,7,8-tetramethyl-OP, 4-phenyl-OP, and OP but does perturb scission by the cuprous complex of 5-phenyl-OP. Organic ligands including intercalating agents and groove binders (e.g., daunomycin, di(amidinophenyl)indole (DAPI), actinomycin D, distamycin, 9-aminoacridine, mithramycin, and chromomycin A3), which bind to free DNA with high affinity, do not form stable ternary complexes with the open-complex. Gel retardation experiments demonstrate that they promote dissociation of the enzyme from the promoter. The greater sensitivity of enzymatic catalysis to inhibitor concentration relative to polymerase binding suggests that these ligands form metastable, catalytically inactive ternary complexes with RNA polymerase and the promoter.