Structural Basis for RCC1 Directed Recruitment of Ran GTPase to Chromatin

RCC1 定向招募 Ran GTP 酶至染色质的结构基础

• 批准号: 8777837
• 负责人: SONG TAN
• 金额: $ 32.52万
• 依托单位: PENNSYLVANIA STATE UNIVERSITY, THE
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-01 至 2018-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8777837
• 关键词: Affect; Architecture; Binding; Binding Proteins; Biochemical; Biology; C-terminal; Cell Nucleus; Cell Separation; Cell division; Cell physiology; Cells; Cellular biology; Characteristics; Chromatin; Chromosome Condensation; Chromosomes; Complex; Crystallization; Cytoplasm; DNA; Data; Dehydration; Drosophila genus; Engineering; Ensure; Epigenetic Process; Eukaryotic Cell; Fluorescent Probes; Funding; GTP Binding; Gene Expression Regulation; Genes; Genetic; Genome; Goals; Guanine; Guanine Nucleotide Exchange Factors; Guanine Nucleotides; Guanosine; Guanosine Triphosphate; Histone H2B; Histones; Human; Location; Malignant Neoplasms; Methods; Mitosis; Mitotic; Molecular; Nuclear Pore Complex; Nucleosome Core Particle; Nucleosomes; Nucleotides; Positioning Attribute; Process; Production; Property; Protein Binding; Proteins; Recruitment Activity; Role; Running; Structure; Surface; System; Work; Yeasts; base; chromatin protein; computerized data processing; daughter cell; genetic regulatory protein; improved; insight; macromolecule; molecular recognition; mutant; nucleocytoplasmic transport; public health relevance; ran GTP-Binding Protein; research study; single molecule; species difference

DESCRIPTION (provided by applicant): 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 nucleotide bound state within the cytoplasm or the nucleus, and by generating a gradient of RanGTP around the chromosomes. This spatial localization of RanGTP in the nucleus is achieved through chromatin bound RCC1 (regulator of chromosome condensation) protein. RCC1 recruits Ran to the nucleosome repeating unit of chromosomes and promotes the exchange of RanGDP for RanGTP, thereby creating a high concentration of RanGTP around chromosomes. The Ran/RCC1/nucleosome complex thus regulates fundamental processes critical for a eukaryotic cell to function properly. Our biochemical studies and crystal structure of the RCC1/nucleosome determined in the last funding period showed how RCC1 binds to the nucleosome. These studies also present new questions about the Ran/RCC1/nucleosome complex, including (a) what regions of Ran interact with RCC1/nucleosome in the Ran/RCC1/nucleosome complex, (b) the role of conformational changes in RCC1/nucleosome upon Ran binding and (c) possible species differences in how Ran and RCC1 interact with the nucleosome. Our goal is therefore to describe how RCC1 and Ran from different species interact with the nucleosome core particle in atomic detail. This proposal focuses on three specific aims: 1. Determine the structure of the Ran/RCC1/nucleosome complex. We will improve single crystals of the Ran/RCC1/nucleosome complex using pre- and post-crystallization strategies to determine the atomic structure of the Ran/RCC1/nucleosome complex. 2. Define how Ran interacts with the nucleosome in Ran/RCC1/nucleosome complexes. We propose biochemical studies to understand which Ran regions interact with the nucleosome in Ran/RCC1/nucleosome complexes from different species, and how these interactions affect Ran's nucleotide exchange activity. 3. Determine role of conformational changes in RCC1/nucleosome & Ran/RCC1/nucleosome complexes. We will use fluorescent probes installed on the nucleosome to study how Ran and RCC1 interact with the nucleosome, and the role of conformational changes in this binding.

描述（由申请人提供）：真核细胞必须能够在其核和细胞质之间方向转运大分子，并通过有丝分裂分裂细胞。这些基本过程是通过在其细胞质或细胞核内的核苷酸结合态中定位小的RAN RAN RAN鸟嘌呤三磷酸酶（GTPase）蛋白来控制的，并通过在染色体周围产生RANGTP的梯度。 RANGTP在细胞核中的空间定位是通过染色质结合的RCC1（染色体凝结）蛋白质实现的。 RCC1募集到染色体的核小体重复单元，并促进RANGDP的RANGTP交换，从而在染色体周围产生高浓度的RangTP。 RAN/RCC1/核小体复合物因此调节真核细胞正常运行至关重要的基本过程。 我们在上一个资金期间确定的RCC1/核小体的生化研究和晶体结构显示了RCC1如何与核小体结合。这些研究还提出了有关RAN/RCC1/核小体复合物的新问题，包括（a）RAN的哪个区域与RAN/RCC1/核小体复合体中的RCC1/核小体相互作用，（b）RCC1/核小体在RAN结合和（C）可能与RAN和RCC1相互作用的物种差异中构象变化的作用。因此，我们的目标是描述在原子细节中与核小体核心粒子相互作用的RCC1和跑步。 该建议重点介绍了三个特定目标：1。确定RAN/RCC1/核小体复合物的结构。我们将使用结晶前和结晶策略来改善RAN/RCC1/核小体复合物的单晶，以确定RAN/RCC1/核小体复合物的原子结构。 2。定义RAN与RAN/RCC1/核小体复合物中的核小体相互作用。我们提出生化研究，以了解哪些RAN区域与来自不同物种的RAN/RCC1/核小体复合物中的核小体相互作用，以及这些相互作用如何影响RAN的核苷酸交换活性。 3。确定构象变化在RCC1/核小体和RAN/RCC1/核小体配合物中的作用。我们将使用核小体上安装的荧光探针研究RAN和RCC1如何与核小体相互作用，以及该结合中构象变化的作用。