Transcription factor regulation of RNA polymerase's torque generation capacity
Transcription factor regulation of RNA polymerase's torque generation capacity
复制标题
RNA聚合酶扭矩产生能力的转录因子调节
DOI：
10.1073/pnas.1807031116
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发表时间：
2019-02-12
影响因子：
11.1
通讯作者：
Wang, Michelle D.
中科院分区：
文献类型：
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作者：
Ma, Jie;Tan, Chuang;Wang, Michelle D.
在转录过程中，RNA聚合酶（RNAP）在DNA易位时缠绕DNA。由此产生的DNA扭转应力可以累积，并且在缺乏调节机制的情况下，成为RNAP延伸的障碍，导致RNAP失速、回溯和转录停滞。在这里，我们研究了转录因子是否以及如何调节扭矩诱导的大肠杆菌RNAP失速和RNAP的扭矩产生能力。使用独特的实时角度光学捕获分析，我们发现RNAP在抵抗扭矩的情况下非常容易发生大范围的回溯。然后，我们研究了GreB存在下的转录，GreB是一种已知的转录因子，可以将RNAP从回溯状态中拯救出来。我们发现GreB极大地抑制了RNAP的回溯，并显著地将RNAP能够产生的扭矩从11.2 pN提高了65%。nm至18.5 pnm。对失速后RNAP的实时位置轨迹进行方差分析，揭示了RNAP回溯和GreB救援的动力学参数。这些结果表明，扭转应力限制转录的主要机制是回溯，转录因子GreB有效地增强了RNAP的扭转能力。这些发现表明转录因子在调节RNAP功能和延伸方面具有更广泛的作用。
During transcription, RNA polymerase (RNAP) supercoils DNA as it translocates. The resulting torsional stress in DNA can accumulate and, in the absence of regulatory mechanisms, becomes a barrier to RNAP elongation, causing RNAP stalling, backtracking, and transcriptional arrest. Here we investigate whether and how a transcription factor may regulate both torque-induced Escherichia coli RNAP stalling and the torque generation capacity of RNAP. Using a unique real-time angular optical trapping assay, we found that RNAP working against a resisting torque was highly prone to extensive backtracking. We then investigated transcription in the presence of GreB, a transcription factor known to rescue RNAP from the backtracked state. We found that GreB greatly suppressed RNAP backtracking and remarkably increased the torque that RNAP was able to generate by 65%, from 11.2 pN.nm to 18.5 pN.nm. Variance analysis of the real-time positional trajectories of RNAP after a stall revealed the kinetic parameters of backtracking and GreB rescue. These results demonstrate that backtracking is the primary mechanism by which torsional stress limits transcription and that the transcription factor GreB effectively enhances the torsional capacity of RNAP. These findings suggest a broader role for transcription factors in regulating RNAP functionality and elongation.