Physics Department, Emory University, Atlanta, GA, USA.
The Center for RNA Biology and Department of Microbiology, The Ohio State University, Columbus, OH, USA.
Nat Commun. 2024 Aug 30;15(1):7520. doi: 10.1038/s41467-024-51603-3.
After an RNA polymerase reaches a terminator, instead of dissociating from the template, it may diffuse along the DNA and recommence RNA synthesis from the previous or a different promoter. Magnetic tweezers were used to monitor such secondary transcription and determine the effects of low forces assisting or opposing translocation, protein roadblocks, and transcription factors. Remarkably, up to 50% of Escherichia coli (E. coli) RNA polymerases diffused along the DNA after termination. Force biased the direction of diffusion (sliding) and the velocity increased rapidly with force up to 0.7 pN and much more slowly thereafter. Sigma factor 70 (σ) likely remained associated with the DNA promoting sliding and enabling re-initiation from promoters in either orientation. However, deletions of the α-C-terminal domains severely limited the ability of RNAP to turn around between successive rounds of transcription. The addition of elongation factor NusG, which competes with σ for binding to RNAP, limited additional rounds of transcription. Surprisingly, sliding RNA polymerases blocked by a DNA-bound lac repressor could slowly re-initiate transcription and were not affected by NusG, suggesting a σ-independent pathway. Low forces effectively biased promoter selection suggesting a prominent role for topological entanglements that affect RNA polymerase translocation.
当 RNA 聚合酶到达终止子时，它可能不会从模板上解离，而是沿着 DNA 扩散，并从前一个或不同的启动子重新开始 RNA 合成。磁镊被用来监测这种二次转录，并确定低力辅助或对抗易位、蛋白质障碍和转录因子的影响。值得注意的是，高达 50%的大肠杆菌（E. coli）RNA 聚合酶在终止后沿着 DNA 扩散。力偏向扩散（滑动）的方向，并且速度随着力的增加而迅速增加，直到 0.7 pN，此后增加得更慢。σ因子 70（σ）可能仍然与 DNA 结合，促进滑动，并能够从任一方向的启动子重新起始。然而，α-C 末端结构域的缺失严重限制了 RNAP 在连续转录循环之间反转的能力。延伸因子 NusG 的添加，它与 σ 竞争与 RNAP 结合，限制了额外的转录循环。令人惊讶的是，被 DNA 结合的 lac 阻遏物阻断的滑动 RNA 聚合酶可以缓慢地重新起始转录，并且不受 NusG 的影响，这表明存在一种不依赖 σ 的途径。低力有效地偏向启动子选择，这表明拓扑缠结在影响 RNA 聚合酶易位方面起着重要作用。
