In eukaryotes, RNA polymerase II (RNApII) transcribes messenger RNA from template DNA. Decades of experiments have identified the proteins needed for transcription activation, initiation complex assembly, and productive elongation. However, the dynamics of recruitment of these proteins to transcription complexes, and of the transitions between these steps, are poorly understood. We used multi-wavelength single-molecule fluorescence microscopy to directly image and quantitate these dynamics in a budding yeast nuclear extract that reconstitutes activator-dependent transcription in vitro. A strong activator (Gal4-VP16) greatly stimulated reversible binding of individual RNApII molecules to template DNA, with no detectable involvement of RNApII-containing condensates. Binding of labeled elongation factor Spt4/5 to DNA typically followed RNApII binding, was NTP-dependent, and was correlated with association of mRNA-binding protein Hek2, demonstrating specificity of Spt4/5 binding to elongation complexes. Quantitative kinetic modeling shows that only a fraction of RNApII binding events are productive and implies a rate-limiting step, probably associated with recruitment of general transcription factors, needed to assemble a transcription-competent pre-initiation complex at the promoter. Spt4/5 association with transcription complexes was slowly reversible, with DNA-bound RNApII molecules sometimes binding and releasing Spt4/5 multiple times. The average Spt4/5 residence time was of similar magnitude to the time required to transcribe an average length yeast gene. These dynamics suggest that a single Spt4/5 molecule remains associated during a typical transcription event, yet can dissociate from RNApII to allow disassembly of abnormally long-lived (i.e., stalled) elongation complexes.

中文翻译：

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激活剂依赖性转录过程中RNA聚合酶II和延伸因子Spt4 / 5募集的动力学。

在真核生物中，RNA聚合酶II（RNApII）从模板DNA转录信使RNA。数十年的实验已经确定了转录激活，起始复合物装配和有效延伸所需的蛋白质。但是，人们对这些蛋白质募集到转录复合体以及这些步骤之间过渡的动力学了解甚少。我们使用多波长单分子荧光显微镜直接成像并量化了萌芽的酵母核提取物中的这些动力学，这些提取物在体外重构了依赖激活物的转录。一个强大的激活剂（Gal4-VP16）极大地刺激了单个RNApII分子与模板DNA的可逆结合，而没有检测到含RNApII的缩合物的参与。标记的延伸因子Spt4 / 5与DNA的结合通常遵循RNApII结合，依赖于NTP，并且与mRNA结合蛋白Hek2的缔合相关，表明Spt4 / 5与延伸复合物结合的特异性。定量动力学建模表明，只有一小部分的RNApII结合事件是有生产能力的，并且暗示了一个限速步骤，可能与募集一般转录因子有关，在启动子上组装有转录能力的预起始复合物是必需的。Spt4 / 5与转录复合物的结合是缓慢可逆的，与DNA结合的RNApII分子有时会多次结合并释放Spt4 / 5。Spt4 / 5的平均停留时间与转录平均长度的酵母基因所需的时间相似。这些动态提示单个Spt4 / 5分子在典型的转录事件中仍保持关联，