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

我们将我们的结构机制研究扩展到真核转录，以确定 保守的和必需的中介体复合物在组装过程中的功能， RNA聚合酶II（pol II）转录前起始复合物（PIC）的分解 以及由pol II启动子引起的趋异转录。基于我们最近 成功开发高效的体外重组转录系统，我们将 利用该系统分离和分析一系列PIC-介体复合物 代表了一系列的状态，从起始到早期延伸，再到可能 在趋异启动子上的功能。我们将探讨潜在的分子机制 这些状态之间的转换，通过结构和生化手段，涉及低温， EM、交联和质谱（XL-MS）以及多波长分析 超离心

We extend our structural mechanistic studies into eukaryotic transcription to determine the functions of the conserved and essential Mediator complex during assembly and disassembly of the RNA polymerase II (pol II) transcription pre-initiation complex (PIC) and in the divergent transcription that arises from pol II promoters. Building on our recent success in developing highly efficient in vitro reconstituted transcription system, we will leverage this system to isolate and dissect a series of PIC-Mediator complexes representing a range of states, from initiation, to early elongation, to complexes that might function on divergent promoters. We will address the molecular mechanisms underlying transitions between these states through structural and biochemical means involving cryo- EM, cross-linking and mass spectrometry (XL-MS), and multi-wavelength analytical ultracentrifugation.