RNA polymerase II (Pol II) and its general transcription factors assemble on the promoters of mRNA genes to form large macromolecular complexes that initiate transcription in a regulated manner. During early transcription, these complexes undergo dynamic rearrangement and disassembly as Pol II moves away from the start site of transcription and transitions into elongation. One step in disassembly is the release of the general transcription factor TFIIB, although the mechanism of release and its relationship to the activity of transcribing Pol II is not understood. We developed a single-molecule fluorescence transcription system to investigate TFIIB release in vitro. Leveraging our ability to distinguish active from inactive complexes, we found that nearly all transcriptionally active complexes release TFIIB during early transcription. Release is not dependent on the contacts TFIIB makes with its recognition element in promoter DNA. We identified two different points in early transcription at which release is triggered, reflecting heterogeneity across the population of actively transcribing complexes. TFIIB releases after both trigger points with similar kinetics, suggesting the rate of release is independent of the molecular transformations that prompt release. Together our data support the model that TFIIB release is important for Pol II to successfully escape the promoter as initiating complexes transition into elongation complexes.

RNA聚合酶II（Pol II）及其一般转录因子在mRNA基因的启动子上组装，形成大分子复合物，以调节方式启动转录。在早期转录过程中，当Pol II远离转录起始位点并转变为延伸序列时，这些复合物会发生动态重排和分解。拆卸的第一步是释放一般转录因子TFIIB，尽管尚不了解释放机理及其与转录Pol II活性的关系。我们开发了一种单分子荧光转录系统来研究TFIIB的体外释放。利用我们区分活性和非活性复合物的能力，我们发现几乎所有转录活性复合物在早期转录过程中都会释放TFIIB。释放不依赖于TFIIB与启动子DNA中其识别元件的接触。我们在早期转录中确定了触发释放的两个不同点，反映了活跃转录复合物群体的异质性。TFIIB在两个具有相似动力学的触发点后释放，表明释放速率与提示释放的分子转化无关。我们的数据一起支持了模型，即TFIIB的释放对于Pol II成功地逃脱启动子非常重要，因为启动复合物过渡为伸长复合物。