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

项目摘要 我们的实验室致力于通过可视化大分子功能的机制理解， 结构、动态和监管互动。为了实现这个目标，我们使用冷冻EM，以及生化 和生物物理测定。我们的研究领域集中在调控分子的表征 微管和人类转录/表观遗传复合体的功能机制。 微管是真核细胞中必需的多聚体，由微管蛋白二聚体组成.动态不稳定性， 由于组装过程与交换的耦合， 和β-微管蛋白中GTP的水解是MT功能的基本性质。许多MT细胞伴侣调节 MT动力学或利用它来执行特定的功能。在过去。在过去的5年里，我们证实， 补充了我们过去的研究，其中使用不可水解的GTP类似物来表征构象 伴随GTP水解的MT变化，现在使用GTP水解微管蛋白突变体。我们还定义了 调节MT组装、动力学和组织的细胞因子的结合和作用模式， 细胞的微管蛋白乙酰化对MT结构和功能的影响。我们将继续这项工作， 增加我们研究的复杂性的中心主题，以使我们更接近调节功能， 微管细胞系统，因为我们还增加了补充我们的主要工具，冷冻EM技术。 基因表达的转录调节对于生长和存活至关重要，并且对于 人体健康其起始涉及RNA聚合酶II（Pol II）连同TFIIA、TFIIB、TFIID、TFIIE、TFIIF和TFIIF。 TFIIH。调控是通过序列特异性激活子或阻遏子、辅因子和染色质来实现的 重塑/修饰复合物。在上一个资助期间，我们定义了人类TFIID的结构[17]， TFIIH[18]，以及其他大的转录辅激活因子，如人佐贺[19]和酵母NuA 4（在 制备）和RSC复合物[20]。我们现在将表征人TIP 60复合物， 转录共激活剂染色质底物，并追求TFIID参与的动态可视化 为了进一步了解这些复合物如何在细胞核中调节基因， 表情 多梳抑制复合物2（Polycomb repressive complex 2，PRC 2）是一种表观遗传基因沉默物，可使组蛋白H3的赖氨酸27甲基化 并且是细胞分化和发育所必需的。在获得人PRC 2的结构后， 辅助因子JARID 2和AEBP 2及其与双核小体的相互作用，我们现在已经定义了PRC 2如何 识别单泛素化核小体，由另一个主要的Polycomb复合物PRC 1产生的底物。 我们现在将进一步表征PRC 2的监管格局，研究其他组蛋白修饰， 不同形式的PRC 2（即，其他辅因子），PRC 2自甲基化的作用，以及PRC 2与RNA的结合。

Project Abstract Our lab is dedicated to the mechanistic understanding of macromolecular function through the visualization of structure, dynamics, and regulatory interactions. Towards that goal, we use cryo-EM, together with biochemical and biophysical assays. Our areas of study are centered on the characterization of the regulatory molecular mechanisms governing the function of microtubules and of human transcription/epigenetic complexes. Microtubules (MTs) are essential polymers in eukaryotic cells s built of -tubulin dimers. Dynamic instability, the switching between growing and shrinking phases due to the coupling of the assembly process to the exchange and hydrolysis of GTP in -tubulin, is an essential property for MT function. Many MT cellular partners modulate MT dynamics or utilize it to carry out specific functions. In the past. In the last 5 years, we confirmed and complemented our past studies, which used non-hydrolyzable GTP analogs to characterize conformational changes in MTs that accompany GTP hydrolysis, now using of GTP-hydrolysis tubulin mutants. We also defined the mode of binding and action of cellular factors that regulate MT assembly, dynamics and organization in the cell and described the effect of tubulin acetylation on MT structure and function. We will continue this work with the central theme of adding complexity to our studies in order to bring us closer to the regulated function of microtubule cellular systems as we also add techniques complementary to our major tool, cryo-EM. Transcriptional regulation of gene expression is critical for growth and survival, and of obvious significance to human health. Its initiation involves RNA polymerase II (Pol II) together with TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH. Regulation is achieved by sequence-specific activators or repressors, co-factors, and chromatin remodeling/modifying complexes. During the last funding period we defined the structures of human TFIID[17] and TFIIH[18], as well as other large transcriptional coactivators like the human SAGA[19], and the yeast NuA4 (in preparation) and RSC complexes[20]. We will now characterize the human TIP60 complex, the binding of transcriptional co-activators to chromatin substrates, and pursue the dynamic visualization of TFIID engagement with promoter DNA to gain further knowledge of how these complexes work in the nucleus to regulate gene expression. Polycomb repressive complex 2 (PRC2) is an epigenetic gene silencer that methylates lysine 27 of histone H3 and is essential for cellular differentiation and development. After obtaining the structure of human PRC2 with its cofactors JARID2 and AEBP2 and its interaction with a di-nucleosome, we have now defined how PRC2 recognizes mono-ubiquitylated nucleosome, the substrate created by the other major Polycomb complex, PRC1. We will now further characterize the regulatory landscape of PRC2 looking into other histone modifications, different forms of PRC2 (i.e., other cofactors), the role of PRC2 auto-methylation, and binding of PRC2 to RNA.