Transcription initiation by RNA polymerase II (RNA Pol II) requires preinitiation complex (PIC) assembly at gene promoters. In the dynamic nucleus, where thousands of promoters are broadly distributed in chromatin, it is unclear how multiple individual components converge on any target to establish the PIC. Here we use live-cell, single-molecule tracking in S. cerevisiae to visualize constrained exploration of the nucleoplasm by PIC components and Mediator’s key role in guiding this process. On chromatin, TFIID/TATA-binding protein (TBP), Mediator, and RNA Pol II instruct assembly of a short-lived PIC, which occurs infrequently but efficiently within a few seconds on average. Moreover, PIC exclusion by nucleosome encroachment underscores regulated promoter accessibility by chromatin remodeling. Thus, coordinated nuclear exploration and recruitment to accessible targets underlies dynamic PIC establishment in yeast. Our study provides a global spatiotemporal model for transcription initiation in live cells.

RNA 聚合酶 II (RNA Pol II) 的转录起始需要在基因启动子处组装预起始复合物 (PIC)。在动态核中，数千个启动子广泛分布在染色质中，目前尚不清楚多个单独组件如何汇聚到任何目标上以建立 PIC。在这里，我们使用酿酒酵母中的活细胞、单分子追踪来可视化 PIC 成分对核质的受限探索以及 Mediator 在指导这一过程中的关键作用。在染色质上，TFIID/TATA 结合蛋白 (TBP)、Mediator 和 RNA Pol II 指示短寿命 PIC 的组装，这种组装很少发生，但平均在几秒钟内有效。此外，核小体侵蚀造成的 PIC 排斥强调了染色质重塑对启动子可及性的调节。因此，协调的核探索和招募到可接近的目标是酵母中动态 PIC 建立的基础。我们的研究提供了活细胞转录起始的全局时空模型。