Beijing National Laboratory for Condensed Matter Physics, Institute of Physics, Chinese Academy of Sciences, Beijing, China.
Songshan Lake Materials Laboratory, Dongguan, Guangdong, China.
Nat Commun. 2024 Jun 15;15(1):5113. doi: 10.1038/s41467-024-49527-z.
Factor-dependent termination uses molecular motors to remodel transcription machineries, but the associated mechanisms, especially in eukaryotes, are poorly understood. Here we use single-molecule fluorescence assays to characterize in real time the composition and the catalytic states of Saccharomyces cerevisiae transcription termination complexes remodeled by Sen1 helicase. We confirm that Sen1 takes the RNA transcript as its substrate and translocates along it by hydrolyzing multiple ATPs to form an intermediate with a stalled RNA polymerase II (Pol II) transcription elongation complex (TEC). We show that this intermediate dissociates upon hydrolysis of a single ATP leading to dissociation of Sen1 and RNA, after which Sen1 remains bound to the RNA. We find that Pol II ends up in a variety of states: dissociating from the DNA substrate, which is facilitated by transcription bubble rewinding, being retained to the DNA substrate, or diffusing along the DNA substrate. Our results provide a complete quantitative framework for understanding the mechanism of Sen1-dependent transcription termination in eukaryotes.
因子依赖的终止利用分子马达来重塑转录机器，但相关的机制，尤其是在真核生物中，还了解甚少。在这里，我们使用单分子荧光分析实时表征由 Sen1 解旋酶重塑的酿酒酵母转录终止复合物的组成和催化状态。我们证实 Sen1 以 RNA 转录本为底物，并通过水解多个 ATP 沿其移动，形成与 RNA 聚合酶 II（Pol II）转录延伸复合物（TEC）停滞的中间产物。我们表明，这种中间产物在单个 ATP 水解后解离，导致 Sen1 和 RNA 解离，之后 Sen1 仍然与 RNA 结合。我们发现 Pol II 最终处于多种状态：从 DNA 底物解离，这得益于转录泡的重绕，与 DNA 底物保持结合，或沿 DNA 底物扩散。我们的结果为理解真核生物中 Sen1 依赖性转录终止的机制提供了一个完整的定量框架。
