Functional and structural studies of the transition from transcription initiation to elongation

这项提案的目标是确定POLII过渡的机制 POL II的启动、延长和重新启动，我们将通过 利用强大的酿酒酵母模型的结构和生化方法 系统。我们开发的用于结构分析的体外转录系统是 基于化学计量比组装的、高活性和均一组分 由30多个多肽组成，包括RNA聚合酶II(PolII)和一般 转录因子(GTF)。关键的是，我们最近成功地优化了这一点 体外重组转录系统，实现双链融化 启动子DNA和RNA从头合成的启动，效率约为100%。我们会 利用这个系统从结构和生化上分离和解剖一系列 POLII复合体代表一系列状态，从起始到早期伸长，再到 可能作为重新引发的支架的复合体。我们将实现这些目标 通过冷冻-EM、交联质谱(XL-MS)和定量XL-MS- 女士。

The goal of this proposal to determine the mechanisms of pol II transitions between initiation, elongation, and re-initiation by pol II, which we will accomplish through structural and biochemical means using the powerful Saccharomyces cerevisiae model system. The in vitro transcription system we have developed for structural analysis is based on stoichiometrically assembled, highly active and homogeneous components comprising over 30 polypeptides, including RNA polymerase II (polII) and the general transcription factors (GTFs). Critically, we have recently succeeded in optimizing this in vitro reconstituted transcription system such that it achieves melting of double-stranded promoter DNA, and initiation of RNA synthesis de novo with ~100% efficiency. We will leverage this system to isolate and dissect, both structurally and biochemically a series of pol II complexes representing a range of states, from initiation, to early elongation, to complexes that might function as scaffolds for reinitiation. We will achieve these aims through Cryo-EM, cross-linking and mass spectrometry (XL-MS), and quantitative XL- MS.