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

项目总结/摘要 我们致力于破译两个基本过程的分子机制： 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。 基因转录是一项复杂的任务，对生长和生存至关重要。虽然入会是最受监管的 转录中的一个步骤，以及它的微调可以在基因表达谱中产生生物体范围的变化， 由于所涉及的分子机制的复杂性，对机制的理解滞后。此外 RNA聚合酶II（Pol II），一般转录因子（GTF：TFIIA、TFIIB、TFIID、TFIIE、TFIIF、TFIIH）） 需要找到转录起始位点（TTS）并将DNA解链和加载到Pol II上。TFIID（~ 1 MDa） 是结合不同核心启动子序列和激活转录所必需的。TFIIH（~450 kDa）是 对于启动子解链和清除启动子所需的Pol II磷酸化以及DNA 修复.真核生物转录机制的结构分析是非常困难的，因为它的稀缺性，穷人， 稳定性和内在的灵活性。我的实验室已经在描述 人TFIID的相互作用，并可视化GTF的人转录前起始复合物（PIC）， 不同的状态，独特地有助于建立转录起始过程的结构框架。 我们现在已做好充分准备，为这一领域作出进一步贡献。我们将定义TFIID的原子结构 和TFIIH，它们落后，并通过添加基因特异性转录因子来构建复杂性， PIC，以便深入了解转录调控的结构基础。 细胞分裂是一个复杂的、高度调节的过程，其中微管（MT）细胞骨架起着中心作用。 作用，作为戏剧性的染色体运动的能量来源，并作为一个支架，促进 在正确的时间和地点发生分子碰撞MT函数的本质是动态不稳定性，这是一个特性， 可以被调节并用于细胞工作。MT是由ab-微管蛋白二聚体自组装而成的 和MT动力学是由于耦合的组装过程中的GTP水解的b-微管蛋白。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细胞伴侣则调节 或者利用动态不稳定性来执行特定功能。通过原子细节的特征， 伴随GTP水解的MTs构象变化，我的实验室已经对结构 MT动力学不稳定性的基础。我们还观察了抗癌药物对MT的结合位点和作用， 并开始定义细胞因子如何与MT表面相互作用，可能影响其结构， 稳定我们现在的主要努力是扩大我们对有丝分裂因子的结合和作用的认识，这些因子调节细胞的增殖， MT动力学和组织，并揭示了真核细胞的基本过程 这将有助于干扰有丝分裂的抗癌剂的改进/开发。

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.