Department of Bionanoscience, Kavli Institute of Nanoscience, Delft University of Technology, 2629 HZ Delft, The Netherlands.
Department of Microbiology, Ohio State University, Columbus, OH 43210, USA.
Cell Rep. 2022 Apr 26;39(4):110749. doi: 10.1016/j.celrep.2022.110749.
Pausing by bacterial RNA polymerase (RNAp) is vital in the recruitment of regulatory factors, RNA folding, and coupled translation. While backtracking and intra-structural isomerization have been proposed to trigger pausing, our mechanistic understanding of backtrack-associated pauses and catalytic recovery remains incomplete. Using high-throughput magnetic tweezers, we examine the Escherichia coli RNAp transcription dynamics over a wide range of forces and NTP concentrations. Dwell-time analysis and stochastic modeling identify, in addition to a short-lived elemental pause, two distinct long-lived backtrack pause states differing in recovery rates. We identify two stochastic sources of transcription heterogeneity: alterations in short-pause frequency that underlies elongation-rate switching, and variations in RNA cleavage rates in long-lived backtrack states. Together with effects of force and Gre factors, we demonstrate that recovery from deep backtracks is governed by intrinsic RNA cleavage rather than diffusional Brownian dynamics. We introduce a consensus mechanistic model that unifies our findings with prior models.
在招募调控因子、RNA 折叠和偶联翻译中，暂停细菌 RNA 聚合酶（RNAp）至关重要。虽然已经提出了回溯和结构内异构化来触发暂停，但我们对与回溯相关的暂停和催化恢复的机制理解仍不完整。我们使用高通量磁镊，在广泛的力和 NTP 浓度范围内检查大肠杆菌 RNAp 的转录动力学。停留时间分析和随机建模确定，除了短暂的基本暂停外，还有两种不同的长寿命回溯暂停状态，它们的恢复速率不同。我们确定了转录异质性的两个随机来源：短暂停频率的变化，这是延伸率切换的基础，以及长寿命回溯状态中 RNA 切割率的变化。结合力和 Gre 因子的影响，我们证明了从深度回溯中恢复是由内在的 RNA 切割而不是扩散布朗动力学控制的。我们引入了一个共识的机制模型，将我们的发现与之前的模型统一起来。
