Wigneshweraraj Siva R, Nechaev Sergei, Severinov Konstantin, Buck Martin
Department of Biological Sciences, Imperial College of Science, Technology and Medicine, Biomedical Sciences Building, Imperial College Road, London SW7 2AZ, UK.
J Mol Biol. 2002 Jun 21;319(5):1067-83. doi: 10.1016/S0022-2836(02)00330-3.
During transcription initiation by DNA-dependent RNA polymerase (RNAP) promoter DNA has to be melted locally to allow the synthesis of RNA transcript. Localized melting of promoter DNA is a target for genetic regulation and is poorly understood at the molecular level. The Escherichia coli RNAP holoenzyme is a six-subunit (alpha(2)betabeta'omegasigma; Esigma) protein complex. The sigma subunit is directly responsible for promoter recognition and contributes to localized DNA melting. Mutations in the beta subunit have profound effects on promoter melting by Esigma70. The sigma54 subunit is a representative of an unrelated class of the sigma subunits. Here, we determined whether mutations in the beta subunit that affect late stages of promoter complex formation by Esigma70 also influence promoter complex formation by the enhancer-dependent Esigma54. Analyses of in vitro defects in promoter complex formation and transcription initiation exhibited by mutant Esigma54 suggest that during promoter complex formation by Esigma54 and Esigma70 a common set of beta subunit sequences is used. Late stages of promoter complex formation and localized melting of promoter DNA by Esigma70 and Esigma54 thus proceed through a common pathway.
在依赖DNA的RNA聚合酶（RNAP）起始转录过程中，启动子DNA必须局部解链以允许RNA转录本的合成。启动子DNA的局部解链是基因调控的一个靶点，在分子水平上人们对此了解甚少。大肠杆菌RNAP全酶是一种六亚基（α₂ββ′ωσ；Eσ）蛋白复合物。σ亚基直接负责启动子识别并有助于局部DNA解链。β亚基中的突变对Eσ⁷⁰介导的启动子解链有深远影响。σ⁵⁴亚基是一类不相关的σ亚基的代表。在此，我们确定了影响Eσ⁷⁰介导的启动子复合物形成后期阶段的β亚基突变是否也会影响依赖增强子的Eσ⁵⁴介导的启动子复合物形成。对突变型Eσ⁵⁴在启动子复合物形成和转录起始方面的体外缺陷分析表明，在Eσ⁵⁴和Eσ⁷⁰介导的启动子复合物形成过程中，使用了一组共同的β亚基序列。因此，Eσ⁷⁰和Eσ⁵⁴介导的启动子复合物形成后期阶段以及启动子DNA的局部解链是通过一条共同途径进行的。
