Structural studies of function and regulation of microtubules and transcriptional gene expression machinery

微管和转录基因表达机制的功能和调节的结构研究

• 批准号: 10231000
• 负责人: Eva Nogales
• 金额: $ 41.44万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-05-01 至 2023-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10231000
• 关键词: Affect; Antineoplastic Agents; Architecture; Binding; Binding Sites; Cell division; Chromosome Segregation; Complex; Coupling; Cryoelectron Microscopy; Cytoskeleton; DNA; DNA Binding; DNA Polymerase II; DNA Repair; DNA-Directed RNA Polymerase; Development; Energy-Generating Resources; Eukaryotic Cell; Gene Expression; Gene Expression Regulation; Genes; Genetic Transcription; Growth; Guanosine Triphosphate; Human; Hydrolysis; Knowledge; Light; Macromolecular Complexes; Microtubules; Mitosis; Mitotic; Modeling; Molecular; Molecular Conformation; Movement; Organism; Paclitaxel; Phosphorylation; Play; Process; Promoter Regions; Property; Regulation; Role; Structure; Surface; TAF1 gene; Time; Transcription Factor TFIIA; Transcription Factor TFIIB; Transcription Initiation; Transcription Initiation Site; Transcription Process; Transcriptional Regulation; Tubulin; Visualization; Work; beta Tubulin; chromosome movement; dimer; flexibility; insight; melting; promoter; scaffold; self assembly; transcription factor; transcription factor TFIIE; transcription factor TFIIF; transcription factor TFIIH

PROJECT SUMMARY/ABSTRACT We are dedicated to deciphering the molecular mechanisms central to two essential processes: transcriptional regulation of gene expression and chromosome segregation by the microtubule (MT) cytoskeleton during cell division. We are using cryo-EM to visualize the molecular players critical to those processes. Gene transcription is a complex task, critical for growth and survival. While initiation is the most regulated step in transcription, and its fine-tuning can produce organism-wide changes in gene expression profiles, a mechanistic understanding lags behind due to the complexity of the molecular machinery involved. In addition to the RNA polymerase II (Pol II), general transcription factors (GTFs: TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH)) are required to find the transcription start site (TTS) and to melt and load the DNA onto Pol II. TFIID (~ 1 MDa) is required for binding to different core promoter sequences and for activated transcription. TFIIH (~450 kDa) is essential for promoter melting and the phosphorylation of Pol II needed to clear the promoter, as well as for DNA repair. Structural analysis of the eukaryotic transcriptional machinery is extremely difficult due to its scarcity, poor stability, and its intrinsic flexibility. My lab has made substantial progress in describing the architecture and DNA interactions of human TFIID, and visualizing the human transcription preinitiation complex (PIC) of GTFs in different states, uniquely contributing to establishing a structural framework for the transcription initiation process. We are now well poised to make further contributions to this field. We will define the atomic structures of TFIID and TFIIH, which lag behind, and build complexity by adding gene-specific transcription factors to our human PICs in order to provide insights into the structural basis of transcriptional regulation. Cell division is a complex, highly regulated process in which the microtubule (MT) cytoskeleton plays a central role, serving as energy source for dramatic chromosomal movements and acting as a scaffold that facilitates molecular encounters at the right time and place. Essential for MT function is dynamic instability, a property that can be both regulated and utilized for cellular work. The MT is built by the self-assembly of ab-tubulin dimers and MT dynamics are due to the coupling of the assembly process to GTP hydrolysis in b-tubulin. Anticancer drugs like taxol stop cell division by interfering with MT dynamics, while many MT cellular partners modulate or utilize dynamic instability to carry out specific functions. By characterizing in atomic detail the conformational changes in MTs that accompany GTP hydrolysis, my lab has shed unique light into the structural basis of MT dynamic instability. We have also visualized the binding site and effect of anticancer drugs on MTs, and started to define how cellular factors interact with the MT surface, potentially affecting its structure and stability. Our main effort now is to extend our knowledge on the binding and effect of mitotic factors that regulate MT dynamics and organization, and shed mechanistic light into a fundamental process for the eukaryotic cell that will contribute to the improvement/development of anticancer agents that interfere with mitosis.

项目摘要/摘要 我们致力于破译两个基本过程中心的分子机制： 微管（MT）细胞骨架对基因表达和染色体分离的转录调节 在细胞分裂期间。我们正在使用Cryo-EM来可视化对这些过程至关重要的分子参与者。 基因转录是一项复杂的任务，对生长和生存至关重要。而启动是最受监管的 转录的一步及其微调可以在基因表达谱的范围内产生变化，A 由于所涉及的分子机械的复杂性，机械理解的滞后。此外 到RNA聚合酶II（POL II），一般转录因子（GTFS：TFIIA，TFIIB，TFIID，TFIIE，TFIIE，TFIIF，TFIIH）））））））） 需要找到转录起始位点（TTS）并将DNA融化并加载到POL II。 TFIID（〜1 MDA） 与不同的核心启动子序列和激活转录结合所必需。 tfiih（〜450 kDa）是 对于清除启动子以及DNA所需的启动子熔化和POL II的磷酸化至关重要 维修。真核转录机械的结构分析由于其稀缺而非常困难，很差 稳定性及其内在灵活性。我的实验室在描述结构和DNA方面取得了重大进展 人类TFIID的相互作用，并可视化GTFS中人类转录前启动复合物（PIC） 不同的状态，有助于建立转录启动过程的结构框架。 现在，我们已经准备好为这一领域做出进一步的贡献。我们将定义TFIID的原子结构 和tfiih，落后于落后，并通过向人类添加基因特异性转录因子来建立复杂性 图片以提供有关转录调控的结构基础的见解。 细胞分裂是一个复杂的，高度调节的过程，其中微管（MT）细胞骨架起中央 角色，充当戏剧性染色体运动的能源，并充当促进的脚手架 分子在正确的时间和地点遇到。 MT功能必不可少的是动态不稳定性，这是一种属性 可以调节和用于细胞工作。 MT是由AB微管蛋白二聚体的自组装建造的 MT动力学是由于组装过程与B-微管蛋白中GTP水解的耦合所致。抗癌 通过干扰MT动力学等药物，例如紫杉醇停止细胞分裂，而许多MT细胞伴侣调节 或利用动态不稳定性来执行特定的功能。通过描述原子细节 GTP水解伴随MT的构象变化，我的实验室将独特的光散发到结构中 MT动态不稳定性的基础。我们还可以观察到抗癌药物对MT的结合位点和影响 并开始定义细胞因子如何与MT表面相互作用，从而可能影响其结构和 稳定。我们现在的主要工作是扩展我们对调节有丝分裂因素的结合和影响的知识 MT动力学和组织，并将机械光放到真核细胞的基本过程中 这将有助于改善干扰有丝分裂的抗癌药物。