RNA polymerase II (RNA Pol II) transcription reconstituted from purified factors suggests pre-initiation complexes (PICs) can assemble by sequential incorporation of factors at the TATA box. However, these basal transcription reactions are generally independent of activators and co-activators. To study PIC assembly under more realistic conditions, we used single-molecule microscopy to visualize factor dynamics during activator-dependent reactions in nuclear extracts. Surprisingly, RNA Pol II, TFIIF, and TFIIE can pre-assemble on enhancer-bound activators before loading into PICs, and multiple RNA Pol II complexes can bind simultaneously to create a localized cluster. Unlike TFIIF and TFIIE, TFIIH binding is singular and dependent on the basal promoter. Activator-tethered factors exhibit dwell times on the order of seconds. In contrast, PICs can persist on the order of minutes in the absence of nucleotide triphosphates, although TFIIE remains unexpectedly dynamic even after TFIIH incorporation. Our kinetic measurements lead to a new branched model for activator-dependent PIC assembly.

从纯化因子重组的 RNA 聚合酶 II (RNA Pol II) 转录表明预起始复合物 (PIC) 可以通过在 TATA 盒中顺序掺入因子来组装。然而，这些基础转录反应通常独立于激活剂和共激活剂。为了在更现实的条件下研究 PIC 组装，我们使用单分子显微镜来观察核提取物中激活剂依赖性反应期间的因子动力学。令人惊讶的是，RNA Pol II、TFIIF 和 TFIIE 可以在加载到 PIC 之前预先组装在增强子结合的激活剂上，并且多个 RNA Pol II 复合物可以同时结合以形成局部簇。与 TFIIF 和 TFIIE 不同，TFIIH 结合是单一的并且依赖于基础启动子。与激活剂有关的因子表现出以秒为单位的停留时间。相比之下，在没有三磷酸核苷酸的情况下，PIC 可以持续数分钟，尽管即使在 TFIIH 掺入后，TFIIE 仍保持出乎意料的动态。我们的动力学测量导致了一个新的依赖于激活剂的 PIC 组装的分支模型。