Efficient gene expression requires RNA polymerase II (RNAPII) to find chromatin targets precisely in space and time. How RNAPII manages this complex diffusive search in three-dimensional nuclear space remains largely unknown. The disordered carboxy-terminal domain (CTD) of RNAPII, which is essential for recruiting transcription-associated proteins, forms phase-separated droplets in vitro, hinting at a potential role in modulating RNAPII dynamics. In the present study, we use single-molecule tracking and spatiotemporal mapping in living yeast to show that the CTD is required for confining RNAPII diffusion within a subnuclear region enriched for active genes, but without apparent phase separation into condensates. Both Mediator and global chromatin organization are required for sustaining RNAPII confinement. Remarkably, truncating the CTD disrupts RNAPII spatial confinement, prolongs target search, diminishes chromatin binding, impairs pre-initiation complex formation and reduces transcription bursting. The present study illuminates the pivotal role of the CTD in driving spatiotemporal confinement of RNAPII for efficient gene expression.

高效的基因表达需要 RNA 聚合酶 II (RNAPII) 在空间和时间上精确地找到染色质靶标。 RNAPII 如何在三维核空间中管理这种复杂的扩散搜索仍然很大程度上未知。 RNAPII 的无序羧基末端结构域 (CTD) 对于招募转录相关蛋白至关重要，在体外形成相分离的液滴，暗示在调节 RNAPII 动力学方面具有潜在作用。在本研究中，我们在活酵母中使用单分子追踪和时空作图来表明，CTD是将RNAPII扩散限制在富含活性基因的亚核区域内所必需的，但没有明显的相分离成凝聚物。维持 RNAPII 限制需要介体和全局染色质组织。值得注意的是，截断 CTD 会破坏 RNAPII 的空间限制，延长靶标搜索时间，减少染色质结合，损害起始前复合物形成并减少转录爆发。本研究阐明了 CTD 在驱动 RNAPII 的时空限制以实现有效基因表达中的关键作用。