Macromolecular Interactions in Yeast Heterochromatin

描述(申请人提供)：真核生物的DNA与核心组蛋白八聚体包装在一起形成核小体阵列。在基因组的某些区域(例如，端粒)，核小体阵列被组装成称为异染色质的特殊结构。包装在异染色质中的基因往往在转录上是沉默的，这将它们与受调控或结构性表达的基因区分开来。在酿酒酵母中，端粒异染色质与由核小体阵列和沉默信息调节蛋白(SIR)蛋白Sir2p、Sir3p和Sir4p组装的特定可遗传核蛋白结构的形成有关。有人提出，SIR蛋白在多亚基SIR复合体中发挥功能，帮助创建转录沉默的异染色质结构域。在目前的提案中，我计划使用定量生化和生物物理方法来表征SIR蛋白的大分子相互作用，无论是在没有核小体阵列的情况下，还是在核小体阵列存在的情况下。具体地说，我建议：(1)表征酵母端粒沉默蛋白在溶液中的自结合和异构体相互作用；(2)表征酵母端粒沉默蛋白与核小体阵列在溶液中的相互作用；(3)利用分析流体力学、电泳和电子显微镜技术表征由酵母沉默蛋白和核小体阵列在体外组装的超分子核蛋白复合体的结构特征。这项研究将首次表征由确定的核小体阵列和纯酵母端粒沉默蛋白在体外形成的核蛋白复合体的组装、稳定性和高阶结构。从这些研究中获得的信息将为理解染色质纤维如何组装成功能专门化的异染色质结构域提供一个框架。此外，为定量研究端粒沉默蛋白复合体而开发的创新方法将普遍适用于千兆道尔顿尺寸范围内的大分子组合的严格溶液分析。

DESCRIPTION (provided by applicant): The DNA of eukaryotes is packaged with core histone octamers to form nucleosomal arrays. In certain regions of the genome (e.g., telomeres), nucleosomal arrays are assembled into specialized structures termed heterochromatin. Genes packaged within heterochromatin tend to be transcriptionally silenced, distinguishing them from the genes which either are regulated or constitutively expressed. In S. cerevisiae, telomeric heterochromatin is associated with formation of a specific heritable nucleoprotein structure assembled from nucleosomal arrays and the silent information regulator (Sir) proteins, Sir2p, Sir3p, and Sir4p. It has been proposed that Sir proteins function in a multi-subunit SIR complex to help create a transcriptionally silenced heterochromatin domain. In the present proposal, I plan to use quantitative biochemical and biophysical approaches to characterize the macromolecular interactions of Sir proteins, both in the absence and presence of nucleosomal arrays. Specifically, I propose to: (1) characterize the self-association and heteromeric interactions of yeast telomeric silencing proteins in solution in vitro, (2) characterize the interaction of yeast telomeric silencing proteins with nucleosomal arrays in solution in vitro, (3) characterize the structural features of the supramolecular nucleoprotein complexes assembled in vitro from yeast silencing proteins and nucleosomal arrays using analytical hydrodynamic, electrophoretic, and electron microscopic techniques. The proposed studies will be the first to characterize the assembly, stability and higher order structures of the nucleoprotein complexes formed in vitro from defined nucleosomal arrays and pure yeast telomeric silencing proteins. The information obtained from these studies will provide a framework for understanding how chromatin fibers are assembled into functionally specialized heterochromatin domains. Additionally, the innovative approach developed to quantitatively study telomeric silencing protein complexes will be generally applicable for rigorous solution analysis of macromolecular assemblages in the giga-dalton size range.