Structural Basis for RCC1 Directed Recruitment of Ran GTPase to Chromatin

项目摘要/摘要 真核细胞必须能够在其核和核之间定向运输大分子 细胞质，并通过有丝分裂来分裂细胞。这些基本过程通过本地化 胞质内GDP或GTP结合状态的小分子鸟苷三磷酸酶(GTPase)蛋白 通过在染色体周围产生RanGTP的梯度，分别在细胞核上产生RanGTP。空间上的 RanGTP在细胞核中的定位是通过染色质结合的RCC1(染色体调节因子)实现的 缩合)蛋白质。RCC1新人跑到染色体上，促进RanGDP换取 RanGTP，从而在染色体周围产生高浓度的RanGTP。 我们目前缺乏对RCC1如何与核小体结合以及RCC1如何与核小体结合的分子理解 新兵跑向核小体，尽管这些相互作用对基本细胞至关重要 流程。因此，我们的总体目标是开发描述RCC1和RAN如何与 核小体核心颗粒。我们的具体目标是： 1.确定RCC1如何通过生化方法与核小体结合。我们将挑战结构性 RCC1如何与核小体相互作用的模型通过下拉、生物层干涉和 荧光共振能量转移实验。 2.确定RCC1/核小体复合体的结构。我们将使用单晶的 我们培养了RCC1/核小体复合体，以确定该复合体的结构。这些结晶学 研究将与小角X射线和中子散射实验相补充，以提供解决方案。 建筑群的结构。 3.确定染色质结合的RCC1如何与RAN结合并激活。我们将对模型进行测试 RAN/RCC1/核小体复合体定向突变对RAN与核小体结合的影响 RCC1/核小体复合体在RCC1和RCC1存在下对RAN核苷酸交换活性的影响 核小体。

Project Summary/Abstract A eukaryotic cell must be able to transport macromolecules directionally between its nucleus and cytoplasm, and to divide the cell through mitosis. These fundamental processes are controlled by localizing the small Ran guanosine triphosphatase (GTPase) protein in its GDP or GTP bound state within the cytoplasm or the nucleus respectively, and by generating a gradient of RanGTP around the chromosomes. The spatial localization of RanGTP in the nucleus is achieved through chromatin bound RCC1 (regulator of chromosomal condensation) protein. RCC1 recruits Ran to the chromosomes and promotes the exchange of RanGDP for RanGTP, thereby creating a high concentration of RanGTP around chromosomes. We currently lack a molecular understanding of how RCC1 binds to the nucleosome and how RCC1 recruits Ran to the nucleosome, despite the critical importance of these interactions for basic cellular processes. Our overall goal is therefore to develop atomic models which describe how RCC1 and Ran bind to the nucleosome core particle. Our specific aims are: 1. Define how RCC1 binds to nucleosomes through biochemical methods. We will challenge structural models for how RCC1 interacts with the nucleosome through pulldown, biolayer interferometry and fluorescence resonance energy transfer experiments. 2. Determine the structure of the RCC1/nucleosome complex. We will use single crystals of the RCC1/nucleosome complex we have grown to determine the structure of the complex. These crystallographic studies will be complemented with small angle X-ray and neutron scattering experiments to provide a solution structure of the complex. 3. Determine how chromatin-bound RCC1 binds to and activates Ran. We will test models for the Ran/RCC1/nucleosome complex by analyzing the effects of directed mutations on binding of Ran to the RCC1/nucleosome complex and on Ran's nucleotide exchange activity in the presence of RCC1 and the nucleosome.