Marr M T, Datwyler S A, Meares C F, Roberts J W
Department of Molecular Biology and Genetics, Biotechnology Building, Cornell University, Ithaca, NY 14853, USA.
Proc Natl Acad Sci U S A. 2001 Jul 31;98(16):8972-8. doi: 10.1073/pnas.161253298.
The structure of an intermediate in the initiation to elongation transition of Escherichia coli RNA polymerase has been visualized through region-specific DNA cleavage by the hydroxyl radical reagent FeBABE. FeBABE was tethered to specific sites of the final sigma(70) subunit and incorporated into two specialized paused elongation complexes that obligatorily retain the final sigma(70) initiation subunit and are targets for modification by lambdoid phage late gene antiterminators. The FeBABE cleavage pattern reveals structures similar to open complex, except for notable changes to region 3 of final sigma(70) that might reflect the presence of stably bound transcript. Binding of the antiterminator protein Q displaces the reactivity of FeBABE conjugated to region 4 of final sigma(70), suggesting that final sigma(70) subunit rearrangement is a step in conversion of RNAP to the antiterminating form.
通过羟基自由基试剂FeBABE进行区域特异性DNA切割,已观察到大肠杆菌RNA聚合酶从起始到延伸转变过程中中间体的结构。FeBABE被连接到最终σ⁷⁰亚基的特定位点,并掺入两个特殊的暂停延伸复合物中,这些复合物必须保留最终的σ⁷⁰起始亚基,并且是λ样噬菌体晚期基因抗终止子修饰的靶点。FeBABE切割模式揭示了与开放复合物相似的结构,除了最终σ⁷⁰的区域3有显著变化,这可能反映了稳定结合转录本的存在。抗终止蛋白Q的结合取代了与最终σ⁷⁰区域4共轭的FeBABE的反应性,表明最终σ⁷⁰亚基重排是RNA聚合酶转化为抗终止形式的一个步骤。