中介体核心复合物调控RNA聚合酶II的结构机制研究

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.

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。

本项目原计划解析裂殖酵母中介体Mediator及其与RNA聚合酶II复合物的冷冻电镜结构，项目执行前一年半，我们已经取得了生化制备、冷冻电镜样品制备和初步的数据收集和三维重构等重要进展，但是受限于高端冷冻电镜机时的紧张，我们没有条件收集大量的高分辨率的冷冻电镜数据；国外同行通过收集超过1万张Titan+K2照片的数据，解析了高分辨率结构并与2017年发表在Nature杂志上。故申请将课题的调整为解析染色质重塑INO80复合体的亚基和模块架构。我们解析染色质重塑INO80复合体的中等分辨率(8~10埃)冷冻电镜三维重构，获得INO80各个亚基的清晰定位，细致分析INO80复合体的亚基组织架构和亚基间的相互作用，构建其亚基和模块的架构模型，阐明各个亚基的生物学功能和复合体组装、功能调控意义；完成Actin/Arp模块的三维结构及其与核小体底物结合的不同重塑状态的三维结构，揭示染色质重塑复合体重塑核小体结构的分子机制（Journal of Molecular Cell Biology，并被选为Research Highlight和杂志封面，同期配有亮点评述）；此外，我们还解析了另一种染色质重塑复合物Swi/Snf的亚纳米分辨率的冷冻电镜结构、模块架构、亚基间相互作用、及其与核小体相互作用方式的结构细节;揭示了Swi/Snf结合核小体、依靠自身结构柔性推动核小体上形成DNA loop的分子机制(Protein & Cell，2018)。这些工作对揭示染色质重塑复合体重塑核小体结构的分子机制，理解基因表达及其调控的分子机制，细胞增殖、发育及分化的机理具有重大意义，并有可能为新型肿瘤治疗药物的开发、干细胞分化与重新编程的改造奠定理论基础。