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：
8910228
负责人：
Michael D Stone
金额：
$ 2.5万
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/8910228
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 biophysical techniques cell age cell growth cofactor comparative flexibility human disease meetings mutant novel novel strategies p65 public health relevance 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.

描述（由申请人提供）：端粒酶核糖核蛋白（RNP）是维持端粒所必需的，端粒是一种特殊的核蛋白结构，可保护真核染色体末端免受异常加工和有害的端到端融合事件的影响。端粒酶使用特殊的催化机制催化端粒 DNA 的持续延伸，该机制需要端粒酶 RNA、端粒酶逆转录酶 (TERT) 和几个其他蛋白质亚基之间具有很强的功能相互依赖性。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域如何协调端粒酶 RNP 组装和催化过程。为了解决与端粒酶结构分析相关的重大挑战，我们将使用多方面的实验策略，结合单分子生物物理技术与计算、生物化学和高分辨率结构方法，研究来自成熟模式生物嗜热四膜虫的端粒酶复合物。在目标 1 中，我们将使用化学 RNA 探测和单分子福斯特共振能量转移 (smFRET) 分别表征端粒酶 RNA 溶液的结构和动力学。这些实验中出现的距离约束将用于指导与 Nikolai Ulyanov (UCSF) 合作的 RNA 结构预测计算。在目标 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 射线晶体学来确定核心端粒酶 RNP 内保守 RNA 和蛋白质结构域的三维组织。这项工作将与 Kathleen Collins (UCB) 和 Harry Noller (UCSC) 合作进行。在目标 3 中，我们将利用一种新型单分子端粒酶结构功能测定来严格评估进行性端粒 DNA 合成过程中端粒酶构象动力学的现有模型。在大多数细胞中，每轮细胞分裂时端粒长度逐渐缩短，为细胞衰老提供分子信号，并调节进入永久性细胞生长停滞。相反，具有高水平增殖能力的细胞（即干细胞）通过端粒酶的酶促作用维持端粒长度。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会导致人类疾病，包括早衰综合症和大多数癌症。因此，端粒酶研究的目标是开发诊断和治疗端粒酶相关疾病的新方法。