Macromolecular Interactions in Yeast Heterochromatin

描述（由申请人提供）：真核生物的DNA与核心组蛋白八聚体一起包装，形成核小体阵列。在基因组的某些区域（例如，端粒），核小体阵列组装成称为异染色质的专门结构。包装在异染色质内的基因往往是转录沉默的，将它们与受调控或组成型表达的基因区分开来。In S.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（Sir）蛋白Sir2p、Sir3p和Sir4p组装的特定可遗传核蛋白结构的形成相关。已经提出Sir蛋白在多亚基SIR复合物中起作用以帮助产生转录沉默的异染色质结构域。在本提案中，我计划使用定量生物化学和生物物理方法来表征的大分子相互作用的爵士蛋白质，无论是在核小体阵列的存在和不存在。具体而言，我建议：（1）在体外表征酵母端粒沉默蛋白在溶液中的自缔合和异聚体相互作用，（2）在体外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）使用分析流体动力学，电泳，和电子显微镜技术。拟议的研究将是第一个表征的组装，稳定性和更高的顺序结构的核蛋白复合物在体外形成的定义的核小体阵列和纯酵母端粒沉默蛋白。从这些研究中获得的信息将提供一个框架，了解染色质纤维是如何组装成功能专门的异染色质结构域。此外，创新的方法开发的定量研究端粒沉默蛋白复合物将普遍适用于严格的解决方案分析的大分子组装在千兆道尔顿的大小范围。

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.