Abstract：
Transcription initiation is the first step in gene expression, and is therefore strongly regulated in all domains of life. The RNA polymerase (RNAP) first associates with the initiation factor $\\sigma$ to form a holoenzyme, which binds, bends and opens the promoter in a succession of reversible states. These states are critical for transcription regulation, but remain poorly understood. Here, we addressed the mechanism of open complex formation by monitoring its assembly/disassembly kinetics on individual consensus lacUV5 promoters using high-throughput single-molecule magnetic tweezers. We probed the key protein-DNA interactions governing the open-complex formation and dissociation pathway by modulating the dynamics at different concentrations of monovalent salts and varying temperatures. Consistent with ensemble studies, we observed that RNAP-promoter open (RPO) complex is a stable, slowly reversible state that is preceded by a kinetically significant open intermediate (RPI), from which the holoenzyme dissociates. A strong anion concentration and type dependence indicates that the RPO stabilization may involve sequence-independent interactions between the DNA and the holoenzyme, driven by a non-Coulombic effect consistent with the non-template DNA strand interacting with $\\sigma$ and the RNAP $\\beta$ subunit. The temperature dependence provides the energy scale of open-complex formation and further supports the existence of additional intermediates.
摘要：
转录起始是基因表达的第一步,因此在生活的所有领域都受到强有力的调节。RNA聚合酶（RNAP）首先与起始因子$\\sigma$结合形成全酶，绑定，以一系列可逆状态弯曲并打开启动子。这些状态对转录调控至关重要，但仍然知之甚少。这里,我们通过使用高通量单分子磁镊子监测其在单个共有lacUV5启动子上的组装/分解动力学,探讨了开放复合物形成的机制.我们通过调节不同浓度的单价盐和不同温度下的动力学，探索了控制开放复合物形成和解离途径的关键蛋白质-DNA相互作用。与合奏研究一致，我们观察到RNAP-启动子开放（RPO）复合物是一个稳定的，缓慢可逆状态，之前是动力学上显著的开放中间体(RPI)，全酶从中解离。强烈的阴离子浓度和类型依赖性表明RPO稳定可能涉及DNA和全酶之间的序列非依赖性相互作用，由非库仑效应驱动，该效应与与$\\sigma$和RNAP$\\beta$亚基相互作用的非模板DNA链一致。温度依赖性提供了开放络合物形成的能量尺度，并进一步支持了其他中间体的存在。
