Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706.
Department of Chemistry, University of Wisconsin-Madison, Madison, WI 53706.
Proc Natl Acad Sci U S A. 2021 Jul 27;118(30). doi: 10.1073/pnas.2021941118.
Transcription initiation is highly regulated by promoter sequence, transcription factors, and ligands. All known transcription inhibitors, an important class of antibiotics, act in initiation. To understand regulation and inhibition, the biophysical mechanisms of formation and stabilization of the "open" promoter complex (OC), of synthesis of a short RNA-DNA hybrid upon nucleotide addition, and of escape of RNA polymerase (RNAP) from the promoter must be understood. We previously found that RNAP forms three different OC with λP promoter DNA. The 37 °C RNAP-λP OC (RP) is very stable. At lower temperatures, RP is less stable and in equilibrium with an intermediate OC (I). Here, we report step-by-step rapid quench-flow kinetic data for initiation and growth of the RNA-DNA hybrid at 25 and 37 °C that yield rate constants for each step of productive nucleotide addition. Analyzed together, with previously published data at 19 °C, our results reveal that I and not RP is the productive initiation complex at all temperatures. From the strong variations of rate constants and activation energies and entropies for individual steps of hybrid extension, we deduce that contacts of RNAP with the bubble strands are disrupted stepwise as the hybrid grows and translocates. Stepwise disruption of RNAP-strand contacts is accompanied by stepwise bubble collapse, base stacking, and duplex formation, as the hybrid extends to a 9-mer prior to disruption of upstream DNA-RNAP contacts and escape of RNAP from the promoter.
转录起始高度受启动子序列、转录因子和配体调控。所有已知的转录抑制剂（一类重要的抗生素）都作用于起始阶段。为了理解调控和抑制机制，必须了解“开放”启动子复合物（OC）的形成和稳定、核苷酸添加时短 RNA-DNA 杂交体的合成以及 RNA 聚合酶（RNAP）从启动子逃脱的生物物理机制。我们之前发现，RNAP 与 λP 启动子 DNA 形成三种不同的 OC。37°C 的 RNAP-λP OC（RP）非常稳定。在较低温度下，RP 不太稳定，处于中间 OC（I）的平衡状态。在这里，我们报告了在 25 和 37°C 下起始和 RNA-DNA 杂交体生长的逐步快速淬火流动力学数据，得出了每个有意义核苷酸添加步骤的速率常数。综合分析，以及之前在 19°C 下发表的数据，我们的结果表明，在所有温度下，I 而不是 RP 是有活性的起始复合物。从杂交体延伸的各个步骤的速率常数和活化能以及熵的强烈变化，我们推断出随着杂交体的生长和迁移，RNAP 与泡状链的接触是逐步被破坏的。RNAP-链接触的逐步破坏伴随着泡状结构的逐步坍塌、碱基堆积和双链形成，当杂交体延伸至 9 个碱基对时，上游 DNA-RNAP 接触被破坏，RNAP 从启动子逃脱。
