Structure and dynamics of the Tetrahymena telomerase ribonucleoprotein

四膜虫端粒酶核糖核蛋白的结构和动力学

基本信息

批准号：
8023853
负责人：
Michael D Stone
金额：
$ 29.26万
依托单位：
UNIVERSITY OF CALIFORNIA SANTA CRUZ
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-12-15 至 2015-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8023853
关键词：
Address Animal Model Binding Biochemical Biological Assay Catalysis Catalytic Domain Cell Aging Cell division Cells Chemicals Chromosomes Collaborations Complex Crystallization Crystallography DNA DNA Primers DNA Sequence Rearrangement DNA biosynthesis Defect Diagnosis Disease Distal Energy Transfer Enzymes Event Fluorescence Resonance Energy Transfer Functional disorder Goals Guide RNA Human Hydroxyl Radical In Vitro Length Malignant Neoplasms Maps Measurement Measures Mediating Medical Modeling Molecular Movement Multienzyme Complexes N-terminal Nucleoproteins Nucleosome Core Particle Pathway interactions Pattern Phylogenetic Analysis Premature aging syndrome Process Property Protein Subunits Proteins Protocols documentation RNA RNA Folding RNA Probes RNA-Protein Interaction Regulation Relative (related person)Research Resolution Rest Ribonucleoproteins Robotics Sequence Analysis Signal Transduction Site Solutions Stem cells Structural Models Structure Structure-Activity Relationship Techniques Telomerase Telomerase RNA Component Telomere Shortening Tertiary Protein Structure Testing Tetrahymena Tetrahymena thermophila Time Transcriptase Work X-Ray Crystallography base cell age cell growth cofactor comparative flexibility human disease meetings mutant novel novel strategies p65 reconstitution research study single molecule software development telomerase reverse transcriptase telomere three dimensional structure

项目摘要

DESCRIPTION (provided by applicant): The telomerase ribonucleoprotein (RNP) is required for maintaining telomeres, the specialized nucleoprotein structures that protect eukaryotic chromosome ends from aberrant processing and deleterious end-to-end fusion events. Telomerase catalyzes the processive extension of telomere DNA using a specialized catalytic mechanism that requires a strong functional interdependence of the telomerase RNA, telomerase reverse transcriptase (TERT), and several additional protein subunits. The primary objective of this proposal is to elucidate how conserved structural domains within telomerase RNA and protein subunits coordinate the processes of telomerase RNP assembly and catalysis. To address the substantial challenges associated with structural analysis of telomerase we will study the telomerase complex from the well-established model organism Tetrahymena thermophila, using a multifaceted experimental strategy that combines single molecule biophysical techniques paired with computational, biochemical, and high-resolution structural approaches. In aim 1, we will use chemical RNA probing and single molecule Forster resonance energy transfer (smFRET) to characterize the telomerase RNA solution structure and dynamics, respectively. Distance constraints that emerge from these experiments will be used to guide RNA structure prediction calculations in collaboration with Nikolai Ulyanov (UCSF). In aim 2, we will determine the three dimensional organization of conserved RNA and protein domains within the core telomerase RNP using targeted-hydroxyl radical probing, smFRET-based structure measurements, and x-ray crystallography. This work will be conducted in collaboration with Kathleen Collins (UCB) and Harry Noller (UCSC). In aim 3, we will exploit a novel single molecule telomerase structure-function assay to critically evaluate existing models for telomerase conformational dynamics during processive telomere DNA synthesis. In most cells, a progressive shortening of telomere length with each round of cell division provides a molecular signal for cell aging and regulates entry into permanent cell growth arrest. In contrast, cells possessing a high level of proliferative capacity (i.e. stem cells) maintain telomere length through the enzymatic action of telomerase. Understanding the molecular mechanism and regulation of telomerase is of direct medical significance because telomerase dysfunction contributes to human disease, including premature aging syndromes and the majority of cancers. Thus, telomerase research is motivated by the goal of developing novel approaches for diagnosing and treating telomerase-associated diseases. PUBLIC HEALTH RELEVANCE: Telomerase is an essential enzyme required for maintaining telomeres, the protective capping structures found at chromosome ends. Telomerase defects arise in the majority (~90%) of human cancers and several premature aging syndromes. Thus, efforts to better understand the mechanism and regulation of telomerase are motivated by the goal of developing novel approaches for diagnosing and treating telomerase-associated diseases. To this end, this proposal aims to illuminate the structural properties of telomerase that underlie its unique cellular activity.

描述（由申请人提供）：维持端粒需要的端粒酶核糖核蛋白（RNP）是保护真核染色体的专门核蛋白结构，这些结构可保护真核染色体末尾，以免异常加工和有害的端到端融合事件。端粒酶使用特殊的催化机制催化端粒DNA的过程扩展，该催化机制需要强大的功能相互依存关系，端粒酶RNA，端粒酶逆转录酶（TERT）和几个其他蛋白质亚基。该提案的主要目的是阐明端粒酶RNA和蛋白质亚基内的保守结构结构域如何协调端粒酶RNP组装和催化的过程。为了解决与端粒酶的结构分析相关的重大挑战，我们将使用多方面的实验策略研究良好的模型有机体四膜植物四氢烷的端粒酶复合物，该策略结合了单个分子生物物理技术与计算，生物化学，生物化学和高分辨率结构方法相结合。在AIM 1中，我们将分别使用化学RNA探测和单分子Forster共振能传递（SMFRET）来表征端粒酶RNA溶液溶液结构和动力学。从这些实验中产生的距离约束将用于与尼古拉·乌利亚诺夫（UCSF）合作指导RNA结构预测计算。在AIM 2中，我们将使用靶向羟基自由基探测，基于SMFRET的结构测量和X射线晶体学，确定核心端酶RNP中保守RNA和蛋白质结构域的三维组织。这项工作将与凯瑟琳·柯林斯（UCB）和哈里·诺勒（UCSC）合作进行。在AIM 3中，我们将利用一种新型的单分子端粒酶结构功能测定法，以批判性地评估端粒型构象动力学在加工端粒DNA合成过程中的现有模型。在大多数细胞中，每一轮细胞分裂的端粒长度逐渐缩短为细胞衰老提供了一个分子信号，并调节进入永久性细胞生长停滞。相反，具有高水平的增殖能力（即干细胞）的细胞通过端粒酶的酶促作用保持端粒长度。了解端粒酶的分子机制和调节具有直接的医学意义，因为端粒酶功能障碍有助于人类疾病，包括早衰综合症和大多数癌症。因此，端粒酶研究的动机是开发出用于诊断和治疗端粒酶相关疾病的新型方法的目标。公共卫生相关性：端粒酶是维持端粒所需的必不可少的酶，即在染色体末端发现的保护性上限结构。端粒酶缺陷出现在大多数人（约90％）的人类癌症和几种过早衰老综合症中。因此，为更好地理解端粒酶的机制和调节的努力是出于开发新型方法来诊断和治疗端粒酶相关疾病的目标。为此，该建议旨在阐明其独特的细胞活性构成端粒酶的结构特性。