Real-time observation of the transition from transcription initiation to elongation of the RNA polymerase
Real-time observation of the transition from transcription initiation to elongation of the RNA polymerase
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DOI：
10.1073/pnas.0906979106
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发表时间：
2009-12-29
影响因子：
11.1
通讯作者：
Patel, Smita S.
中科院分区：
文献类型：
--
作者：
Tang, Guo-Qing;Roy, Rahul;Patel, Smita S.
RNA聚合酶（RNAP）从起始到延伸的转变是转录的重要阶段，其通常限制全长RNA的产生。由于在快速和真实的时间中监测瞬时和异质转录复合物的亚群的挑战，关于RNAP转换动力学和决定转换速率的步骤知之甚少。在这里，我们已经解剖了完整的转录起始途径T7 RNAP通过使用RNA合成的动力学建模，并通过确定在每个RNA聚合步骤的起始（IC）延长（EC）过渡动力学，使用单分子和停流FRET方法。我们发现，IC转换为EC在T7 RNAP共识启动子仅发生后8- 12-nt的合成，和12-nt的合成代表了一个关键时刻，在转录起始途径时，EC的形成是最有效的。我们发现，转录起始的缓慢步骤，包括DNA scrunching/RNAP启动子旋转的变化在5- 8-nt的合成，而不是主要的构象变化，决定了EC的形成在T7 RNAP的整体速度和代表的关键步骤，调节全长RNA的合成。
The transition from initiation to elongation of the RNA polymerase (RNAP) is an important stage of transcription that often limits the production of the full-length RNA. Little is known about the RNAP transition kinetics and the steps that dictate the transition rate, because of the challenge in monitoring subpopulations of the transient and heterogeneous transcribing complexes in rapid and real time. Here, we have dissected the complete transcription initiation pathway of T7 RNAP by using kinetic modeling of RNA synthesis and by determining the initiation (IC) to elongation (EC) transition kinetics at each RNA polymerization step using single-molecule and stopped-flow FRET methods. We show that the conversion of IC to EC in T7 RNAP consensus promoter occurs only after 8- to 12-nt synthesis, and the 12-nt synthesis represents a critical juncture in the transcriptional initiation pathway when EC formation is most efficient. We show that the slow steps of transcription initiation, including DNA scrunching/RNAP-promoter rotational changes during 5- to 8-nt synthesis, not the major conformational changes, dictate the overall rate of EC formation in T7 RNAP and represent key steps that regulate the synthesis of full-length RNA.