A comparative structural study of ATP-dependent chromatin remodeling complexes

描述（由申请人提供）：真核细胞已经解决了基因组包装问题，将染色质（由DNA和组蛋白组成的核蛋白纤维）与修饰它并调节其动力学的因子结合起来。其中包括ATP依赖性染色质重塑复合物或“重塑物”，即使用ATP水解来非共价改变核小体结构的大型且保守的多亚基组装体。重塑剂可以分为四个家族，并且在组成和它们在体内和体外产生的产物方面都不同。染色质重塑的机制是什么？不同的产品是如何产生的？这些问题在很大程度上仍然没有答案，这是由于重塑者的复杂性和缺乏结构信息的组合，从而为积累的生物化学和遗传数据提供背景。目前的机制模型很难，如果不是不可能的话，单独用生化方法进行测试。 冷冻电子显微镜（Cryo-EM）非常适合于分析重塑和什么似乎是他们的内在构象灵活性。虽然回答许多机械问题所需的高分辨率是一个具有挑战性的长期目标，但更容易实现的低分辨率结构可以为我们的建模提供重要的约束，并深入了解重塑者的专业化。我们将运用我们的专业知识，在电子显微镜的大型，不对称和异质大分子组装开始两个重塑具有非常不同的活动：组蛋白八聚体滑动和组蛋白二聚体交换的比较结构研究。我们的模型系统，都是~ 1 MDa，是S。酿酒酵母重塑蛋白RSC，其滑动八聚体，和SWR 1，其将H2 A. Z/H2 B二聚体交换为天然H2 A/H2 B。 在目标1中，我们将获得SWR 1复合物本身和与核小体结合的重建。在目标2中，我们将获得与核小体结合的RSC（RSCsub）的重塑能力4-亚基亚复合物的结构，并将我们目前的RSC结构用冷冻水合样品细化到更高的分辨率。在目标3中，我们将通过绘制SWR 1和RSC/RSCsub中几个关键亚基的位置，从目标1和2中获得的结构中提取生物信息。我们已经选择了目标，这将最大限度地提高我们可以获得的机械洞察力，即使在中等分辨率下，我们希望在此资助期内获得。RSC的靶标也被设计成允许我们将RSC亚核小体结构对接到完整的RSC中。目标3的一个重要组成部分是开发用于标记冷冻水合样品中亚基的新方法。

DESCRIPTION (provided by applicant): Eukaryotic cells have solved the genome-packaging problem combining chromatin, the nucleoprotein fiber consisting of DNA and histones, with factors that modify it and regulate its dynamics. Among them are the ATP- dependent chromatin remodeling complexes or "remodelers", large and conserved multi-subunit assemblies that use ATP hydrolysis to non-covalently alter nucleosome structure. Remodelers can be classified into four families and differ both in composition and the products they generate both in vivo and in vitro. What is the mechanism of chromatin remodeling? How are different products generated? These questions remain unanswered due, to a large extent, to a combination of the remodelers' complexity and a paucity of structural information to provide a context for the accumulated biochemical and genetic data. Current mechanistic models are difficult, if not impossible, to test with biochemical approaches alone. Cryo-electron microscopy (Cryo-EM) is ideally suited for the analysis of remodelers and of what appears to be their intrinsic conformational flexibility. While the high resolution required to answer many mechanistic questions is a challenging long-term goal, more easily achievable lower resolution structures can provide important constraints to our modeling as well as insights into remodeler specialization. We will apply our expertise in electron microscopy of large, asymmetric and heterogeneous macromolecular assemblies to begin a comparative structural study of two remodelers with very different activities: histone octamer sliding and histone dimer exchange. Our model systems, both ~1MDa, are the S. cerevisiae remodelers RSC, which slides octamers, and SWR1, which exchanges H2A.Z/H2B dimers for the native H2A/H2B. In Aim 1 we will obtain reconstructions of the SWR1 complex both by itself and bound to a nucleosome. In Aim 2 we will obtain the structure of a remodeling-competent 4-subunit subcomplex of RSC (RSCsub) bound to a nucleosome and will refine our current RSC structure to higher resolution with frozen-hydrated samples. In Aim 3 we will extract biological information from the structures obtained in Aims 1 and 2 by mapping the location of a few key subunits in SWR1 and RSC/RSCsub. We have selected targets that will maximize the mechanistic insight we can gain even at the medium-resolution we expect to obtain within this funding period. The targets for RSC are also designed to allow us to dock the RSCsub-nucleosome structure into full RSC. An important component of Aim 3 is the development of novel methods for labeling subunits in frozen-hydrated samples.