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Temperature effects on RNA polymerase initiation kinetics reveal which open complex initiates and that bubble collapse is stepwise [Biochemistry]
Proceedings of the National Academy of Sciences of the United States of America ( IF 11.1 ) Pub Date : 2021-07-27 , DOI: 10.1073/pnas.2021941118
Dylan M Plaskon 1 , Kate L Henderson 1 , Lindsey C Felth 1 , Cristen M Molzahn 1 , Claire Evensen 1 , Sarah Dyke 1 , Irina A Shkel 1, 2 , M Thomas Record 2, 3
Affiliation  

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 λPR promoter DNA. The 37 °C RNAP-λPR OC (RPO) is very stable. At lower temperatures, RPO is less stable and in equilibrium with an intermediate OC (I3). 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 I3 and not RPO 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.



中文翻译:

温度对 RNA 聚合酶起始动力学的影响揭示了哪个开放复合物起始,并且气泡破裂是逐步的 [生物化学]

转录起始受启动子序列、转录因子和配体的高度调控。所有已知的转录抑制剂,一类重要的抗生素,都在起始中起作用。为了理解调控和抑制,“开放”启动子复合物 (OC) 的形成和稳定、核苷酸添加时短 RNA-DNA 杂合体的合成以及 RNA 聚合酶 (RNAP) 从启动子逃逸的生物物理机制必须被理解。我们之前发现 RNAP 与 λPR 启动子 DNA 形成三种不同的OC。37 °C RNAP-λP R OC (RP O ) 非常稳定。在较低温度下,RP O不太稳定并与中间体 OC (I 3)。在这里,我们报告了 RNA-DNA 杂合体在 25 和 37°C 下启动和生长的逐步快速淬火流动力学数据,这些数据在生产性核苷酸添加的每个步骤中产生速率常数。与之前发布的 19 °C 数据一起分析,我们的结果显示 I 3而不是 RP O是所有温度下的生产引发复合物。从混合延伸的各个步骤的速率常数、活化能和熵的强烈变化中,我们推断随着混合体的生长和易位,RNAP 与气泡链的接触会逐步中断。RNAP 链接触的逐步破坏伴随着逐步气泡破裂、碱基堆积和双链体形成,因为在上游 DNA-RNAP 接触破坏和 RNAP 从启动子逃逸之前,杂交体延伸到 9 聚体。

更新日期:2021-07-22
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