中介体被称为转录中央控制器，解析中介体、中介体-RNA聚合酶II全酶复合物的精细结构对于揭示转录调控的分子机制至关重要。申请人最近完成了第一个中介体多种构象状态的三维重构；“庖丁解牛”解析了首个中介体“头部+中部”模块组合的结构；清晰划定了各个模块的边界，首次建立了正确的中介体模块化结构。此外，相继解析多个物种的中介体发现裂殖酵母（ S. pombe）中介体仅含有功能核心（头部/中部模块，缺少高度动态的尾部模块）结构上相对稳定，非常适合解析中介体和全酶复合物的高分辨率结构；计划利用冷冻电镜构建裂殖酵母中介体和全酶的准原子模型，期望从结构上阐明中介体调控RNA聚合酶II转录活性的分子机制。

Mediator, a large complex highly conserved throughout eukaryotes, conveys regulatory signals that influence the activity of the RNA polymerase II (RNAPII). However, the detailed architecture of Mediator, the precise mode of interaction between Mediator and RNAPII, and the mechanism of regulation by Mediator remain largely elusive. Recently, I have determined the first Mediator EM reconstructions in different conformational states and the first 3D structure of the Head+Middle modules, which permitted an unambiguous assignment of the three modules and completely redefined the modular organization of core Mediator. Moreover, I characterized the Mediator from several species and found the Mediator from the fission yeast S. pombe contains only the Head and Middle modules together forming a functional core to bind RNAPII. Since the highly mobile Tail module is absent, the structure of S. pombe Mediator is much more stable, which is promising for cryo reconstruction with significantly higher accuracy and resolution. In this grant application, I propose a series of focused studies that will generate 8~10 Å cryo-EM reconstructions of Mediator and Mediator-RNAPII holoenzyme from S. pombe and build the near-atomic structural models, providing information about Mediator’s detailed architecture and functional interaction with RNAPII and testing fundamental hypotheses about the mechanism of transcription regulation by Mediator. The preliminary results that we present suggest that the resolution of cryo-EM reconstruction of Mediator and holoenzyme could be substantially improved and that completion of our goals will provide information that will significantly contribute to deciphering the mechanism of Mediator on transcriptional regulation.