Qian Jin, Cartee Allison G, Xu Wenxuan, Yan Yan, Wang Bing, Artsimovitch Irina, Dunlap David, Finzi Laura
bioRxiv. 2023 Jun 26:2023.01.04.522798. doi: 10.1101/2023.01.04.522798.
RNA polymerases (RNAPs) must transit through protein roadblocks to produce full-length RNAs. Here we report real-time measurements of Escherichia coli (E. coli) RNAP passage through different barriers. As intuitively expected, assisting forces facilitated, and opposing forces hindered, RNAP passage through LacI bound to natural operator sites. Force-dependent differences were significant at magnitudes as low as 0.2 pN and were abolished in the presence of GreA, which rescues backtracked RNAP. In stark contrast, opposing forces promoted passage when the rate of backtracking was comparable to, or faster than the rate of dissociation of the roadblock, particularly in the presence of GreA. Our experiments and simulations indicate that RNAP may transit after roadblocks dissociate, or undergo cycles of backtracking, recovery, and ramming into roadblocks to pass through. We propose that such reciprocating motion also enables RNAP to break protein-DNA contacts holding RNAP back during promoter escape and RNA chain elongation, facilitating productive transcription in vivo.
RNA聚合酶（RNAPs）必须穿过蛋白质障碍才能产生全长RNA。在此，我们报告了大肠杆菌（E. coli）RNAP穿过不同障碍的实时测量结果。正如直观预期的那样，助力促进了RNAP穿过与天然操纵子位点结合的LacI，而阻力则阻碍了RNAP的通过。在低至0.2皮牛顿的力大小下，力依赖性差异就很显著，并且在存在GreA的情况下差异消失，GreA可拯救回溯的RNAP。与此形成鲜明对比的是，当回溯速率与障碍解离速率相当或更快时，尤其是在存在GreA的情况下，阻力会促进通过。我们的实验和模拟表明，RNAP可能在障碍解离后通过，或者经历回溯、恢复以及撞击障碍以通过的循环过程。我们提出，这种往复运动还使RNAP能够打破在启动子逃逸和RNA链延伸过程中阻碍RNAP的蛋白质 - DNA接触，从而促进体内的有效转录。
