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

本提案的目标是确定pol II在 通过PolII引发、延伸和再引发，我们将通过 使用强大的酿酒酵母模型的结构和生化手段 系统我们开发的用于结构分析的体外转录系统是 基于化学计量组装、高活性和均质组分 包含超过30种多肽，包括RNA聚合酶II（polII）和一般的 转录因子（GTF）。至关重要的是，我们最近成功地优化了这一点， 体外重建的转录系统，使得它实现双链的解链， 启动子DNA，并以~100%的效率从头开始RNA合成。我们将 利用这个系统从结构上和生物化学上分离和剖析一系列 pol II复合物代表一系列状态，从起始到早期延伸， 复合物可能作为支架重新启动。我们将实现这些目标 通过Cryo-EM，交联和质谱（XL-MS），和定量XL- 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.