Wigneshweraraj Siva R, Burrows Patricia C, Nechaev Sergei, Zenkin Nikolay, Severinov Konstantin, Buck Martin
Department of Biological Sciences, Imperial College London, London SW7 2AZ, UK.
EMBO J. 2004 Oct 27;23(21):4264-74. doi: 10.1038/sj.emboj.7600407. Epub 2004 Oct 7.
We used bacteriophage T7-encoded transcription inhibitor gene protein 2 (gp2) as a probe to study the contribution of the Escherichia coli RNA polymerase (RNAP) beta' subunit jaw domain--the site of gp2 binding--to activator and ATP hydrolysis-dependent open complex formation by the sigma(54)-RNAP. We show that, unlike sigma(70)-dependent transcription, activated transcription by sigma(54)-RNAP is resistant to gp2. In contrast, activator and ATP hydrolysis-independent transcription by sigma(54)-RNAP is highly sensitive to gp2. We provide evidence that an activator- and ATP hydrolysis-dependent conformational change involving the beta' jaw domain and promoter DNA is the basis for gp2-resistant transcription by sigma(54)-RNAP. Our results establish that accessory factors bound to the upstream face of the RNAP, communicate with the beta' jaw domain, and that such communication is subjected to regulation.
我们使用噬菌体T7编码的转录抑制基因蛋白2（gp2）作为探针，来研究大肠杆菌RNA聚合酶（RNAP）β'亚基颌结构域（gp2的结合位点）对σ⁵⁴-RNAP激活剂和ATP水解依赖性开放复合物形成的贡献。我们发现，与依赖σ⁷⁰的转录不同，σ⁵⁴-RNAP的激活转录对gp2具有抗性。相反，σ⁵⁴-RNAP的激活剂和ATP水解非依赖性转录对gp2高度敏感。我们提供的证据表明，涉及β'颌结构域和启动子DNA的激活剂和ATP水解依赖性构象变化是σ⁵⁴-RNAP对gp2抗性转录的基础。我们的结果表明，与RNAP上游面结合的辅助因子与β'颌结构域相互作用，并且这种相互作用受到调控。
