本论文包括两部分内容：TFIID复合物组装与启动子识别机制的研究以及人源mTORC2复合物的冷冻电镜结构研究。 在本论文的第一部分，我们研究了TFIID复合物的组装及其识别启动子的分子机制。RNA聚合酶Ⅱ(Pol Ⅱ)的转录起始过程是真核生物细胞内基因差异性表达的关键调控点之一。Pol Ⅱ介导的基因转录起始过程需要多个通用转录因子(GTFs)和Pol Ⅱ的共同参与，它们按照一定的先后顺序依次被招募至基因的核心启动子区组装形成转录前起始复合物(preinitiation complex，PIC)，起始基因的转录。TFIID是第一个结合到基因启动子上并触发PIC组装的通用转录因子，能够识别多种核心启动子元件，被认为是主要的核心启动子结合因子。尽管在体外单独的TBP蛋白也能够组装形成具有基本转录活性的PIC，但在后生动物的转录系统中，仅由完整的TFIID复合物所组装形成的PIC才能够应答转录激活效应。此外，TFIID还可以作为共激活因子(coactivator)通过与转录激活因子(activators)和表观遗传修饰相互作用调控基因的转录。虽然TFIID在真核生物基因转录中扮演着如此关键的角色，但是其详细的组装及调控的分子机制目前尚不清楚。 TFIID全复合物由lobe A、lobe B和lobe C三部分组成。我们利用冷冻电镜技术分别解析了分辨率为3.4Å、3.0Å和4.0Å的TFIID复合物中lobe A、lobe B以及lobe C部分的冷冻电镜结构，以及五种不同状态的TFIID和TFIID-promoter复合物的冷冻电镜结构。结构显示，lobe B和lobe A呈现相似的构象，分别由含有WD40结构域的TAF5及核小体样(nucleosome-like)的组蛋白折叠结构域(HFD)-六聚体(TAF4-TAF12、TAF6-TAF9和TAF8-TAF10)或HFD-八聚体(TAF4-TAF12、TAF6-TAF9、TAF3-TAF10和TAF11-TAF13)所组成。 lobe B和lobe A中存在两个关键的转换区域能够抑制TAF11-TAF13与TAF8特异性的lobe B的组装并促进TAF11-TAF13与TAF3特异性的lobe A的组装。 TAF1-TAF7和TAF2作为两个独立的启动子识别模块，能够独立且灵活地识别多种类型的启动子。有趣的是，对于不含有TATA框的人类PUMA基因启动子，当TFIID结合后，该启动子上游区域TBP蛋白结合处仍会发生弯曲，暗示TFIID结合启动子后会以一种保守的构象状态来进一步介导基因转录的起始。我们的研究为理解TFIID复合物的组装及其识别不同类型启动子的分子机制提供了结构基础。 在本论文的第二部分中我们研究了人源mTORC2复合物的结构。哺乳动物雷帕霉素靶标蛋白(mammalian target of TOR，mTOR)是一个在进化上保守的丝氨酸/苏氨酸蛋白激酶，在哺乳动物体内，mTOR会与不同的蛋白组装形成两种不同的复合物：对雷帕霉素敏感的mTOR复合物1(mTORC1)和对雷帕霉素不敏感的mTOR复合物2(mTORC2)。其中，mTORC2核心复合物由mTOR、mLST8以及两种mTORC2特异性的蛋白组分Rictor和mSin1所组成。在体内， mTORC2会磷酸化AGC蛋白激酶家族成员(包括AKT、PKC和SGK1)调控细胞的增殖。研究显示，mTORC2信号通路的异常会导致肿瘤的发生，是潜在的抗癌药物靶标。在这部分的研究工作中，我们利用冷冻电镜单颗粒重构技术解析了分辨率为4.9Å的mTORC2核心复合物结构，同时结合化学交联质谱和免疫共沉淀的方法揭示了mTORC2各组分分子间的相互作用关系。结构显示， mTORC2整体呈现二次对称的中空棱形，两个mTOR蛋白形成二聚体位于中央，介导复合物的组装。Rictor的N端由大量的螺旋重复簇(helical repeats clusters)组成，与mTOR蛋白之间存在多个相互作用面。mSin1位于mTOR蛋白的FRB结构域和催化口袋附近。从结构中来看，mTORC2中Rictor和mSin1两个蛋白的存在对FKBP12-雷帕霉素与mTOR蛋白的结合产生了空间位阻，这种空间阻碍阻止了二者的相互作用，使得mTORC2的活性不受雷帕霉素所抑制。此外，结构比对显示，mTORC2整体与mTORC1采取相似的构象，其特异性组分Rictor和Raptor(mTORC1特异性组分)与mTOR蛋白之间存在着相同的相互作用方式和位点，暗示二者与mTOR的结合是相互排斥的，揭示了两个复合物的不同组装方式。我们的研究为理解mTORC2的分子组装方式和调控机制提供了结构研究基础。 关键词：冷冻透射电子显微镜；结构生物学；TFIID；启动子识别；mTORC2 中图分类号：Q5

 This thesis contains two parts: the structural insights into assembly of and promoter recognition by TFIID holocomplex and the cryo-EM structural studies of human mTOR complex 2. In the first part of this thesis, we studied the molecular mechanism for TFIID complex assembly and promoter recognition. The initiation of gene transcription by RNA polymerase Ⅱ (Pol Ⅱ) is one of the most critical regulatory point for differential gene expression in eukaryotic cells. The pol Ⅱ-mediated transcription requires a sequential recruitment of Pol Ⅱ and general transcrpiton factors (GTFs) to the core promoters for assembly of pre-initiation complex (PIC). TFIID is the first GTF that binds gene promoters to trigger the PIC formation. TFIID binds various core promoter elements and is believed to be the primary core promoter binding factor. Although TBP is sufficient for recognition of core promoter and assembly of a functional PIC with basal transcription activity, transcriptional acrivation in metazoan systems was observed only when the PIC was assembled with the TFIID complex. TFIID also functions as a coactivator through interacting with transcriptional activators and reading epigenetic marks. Despite its critical role in eukaryotic gene transcription, understanding the complex assembly and structure-function relationship of TFIID are largely limited. TFIID holocomplex contains lobe A, lobe B and lobe C. We determined the cryo-EM structures of lobe A, lobe B, and part of lobe C subcomplexes at 3.4?, 3.0?, and 4.0? resolution, respectively, and structures of TFIID-promoter holocomplexes in five distinct states. The lobes B and A adopt similar fold and consist of a WD40-containing TAF5 associated with nucleosome-like histone fold hexamer (TAF4-TAF12, TAF6-TAF9, TAF8-TAF10) and octamer (TAF4-TAF12, TAF6-TAF9, TAF3-TAF10, TAF11-TAF13), respectively. Two switch regions prohibit TAF11-TAF13 association with TAF8-specific lobe B but facilitate TAF3-specific lobe A assembly. TAF1-TAF7 and TAF2 serve as independent promoter-binding modules to facilitate recognizing various type of promoters. Unexpectedly, although lacking TATA box, the human PUMA promoter would undergo bending upon binding TFIID, indicating a conserved conformation of TFIID-bound promoters for transcriptional initiation. Our study provides structural basis for understanding the assembly of TFIID holocomplexes and recognition of varioustype of core promoters. In the second part of this thesis, we studied the cryo-EM structure of human mTOR complex 2.The mammalian target of rapamycin (mTOR) is a giant Ser/Thr kinase and is evolutionally conserved in eukayotes from yeast to human. mTOR functions as a master regulator in controlling various cellular processes through forming two distinct multisubunit protein complexes: a rapamycin-sensitive mTOR complex1 (mTORC1) and a rapamycin-insensitive mTOR complex 2 (mTORC2). The functional core complex consists of mTOR, mLST8, and two mTORC2 specific components, Rictor and mSin1. In mammalian cell, the mTOR complex 2 (mTORC2) plays an essential role in regulating cell proliferation through phosphorylating AGC protein kinase family members, including AKT, PKC, and SGK1. Aberrant mTORC2 signaling has been shown to involve tumorigenesis and mTORC2 may serve as a potential anti-cancer durg target. Here, we investigated the intermolecular interactions within mTORC2 complex and determined its cryo-electron microscopy structure at 4.9 A resolution. The structure reveals a hollow rhombohedral fold with a 2-fold symmetry. The dimerized mTOR serves as a scaffold for the complex assembly. The N-terminal half of Rictor is composed of helical repeats clusters and binds to mTOR through multiple contacts. mSin1 is located close to the FRB domain and catalytic cavity of mTOR. Rictor and mSin1 together generate steric hindrance to inhibit binding of FKBP12-rapamycin to mTOR, which reveals the mechanism for rapamycin insensitivity of mTORC2. Structural comparison shows that binding of Rictor and Raptor (mTORC1 specific component) to mTOR is mutually exclusive. Our study provides a basis to understand the assembly of mTORC2 and a framework to further characterize the regulatory mechanism of mTORC2 pathway. Key Words: Cryo-EM; Structure; TFIID; promoter recognition; mTORC2 CLC code:Q5