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: 10399598
• 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 2023-08-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10399598
• 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的核心分子机制： 微管细胞骨架对基因表达和染色体分离的转录调控 在细胞分裂过程中。我们正在使用冷冻-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。而入会是最受监管的 在转录过程中，它的微调可以在整个生物体范围内产生基因表达谱的变化， 由于涉及的分子机制的复杂性，机械论的理解滞后了。此外 对于RNA聚合酶II(POL II)、通用转录因子(GTF：TFIIA、TFIIB、TFIID、TFIIE、TFIIF、TFIIH)) 需要找到转录起始点(TTS)，并将DNA熔化并加载到Pol II上。 是与不同核心启动子序列结合和激活转录所必需的。TFIIH(~450 kDa)是 对于启动子熔化和Pol II的磷酸化是必不可少的，需要清除启动子和DNA 修理。真核转录机制的结构分析是极其困难的，因为它是稀缺的，差的 稳定性及其内在的灵活性。我的实验室在描述体系结构和DNA方面取得了实质性进展 人TFIID的相互作用和人转录预起始复合体(PIC)的可视化 不同的状态，独特地有助于建立转录起始过程的结构框架。 我们现在已经做好了为这一领域做出进一步贡献的准备。我们将定义TFIID的原子结构 和TFIIH，它们落后了，通过向我们的人类添加基因特异的转录因子来建立复杂性 PICS，以提供对转录调控的结构基础的见解。 细胞分裂是一个复杂的、高度调控的过程，微管(MT)细胞骨架在其中发挥核心作用 角色，作为戏剧性染色体运动的能量来源，并充当促进 分子在正确的时间和地点相遇。MT功能的关键是动态不稳定性，这是一种 既可以被调节，也可以被用于细胞工作。MT是由ab-微管蛋白二聚体自组装而成的 MT的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 像紫杉醇这样的药物通过干扰MT动力学来阻止细胞分裂，而许多MT细胞伙伴则调节 或者利用动态不稳定性来执行特定的功能。通过对原子细节的描述， 伴随着GTP水解的MT的构象变化，我的实验室已经为结构提供了独特的线索 大地电磁动力不稳定的基础。我们还可视化了抗癌药物在MTS上的结合部位和作用， 并开始定义细胞因子如何与MT表面相互作用，潜在地影响其结构和 稳定性。我们现在的主要工作是扩展我们对调节有丝分裂因子的结合和影响的知识。 MT的动力学和组织，并揭示了真核细胞的基本过程 这将有助于改善/发展干扰有丝分裂的抗癌药物。