Structure and Dynamics of the Telomerase Ribonucleoprotein

端粒酶核糖核蛋白的结构和动力学

基本信息

批准号：
8962176
负责人：
Michael D Stone
金额：
$ 33.73万
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 2019-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8962176
关键词：
Address Binding Biochemical Biological Assay Biological Models Catalysis Cells Chromosomes Collaborations Complex Computer Simulation Crystallography DNA DNA Binding DNA Structure DNA biosynthesis Defect Deterioration Diagnosis Disease Energy Transfer Enzymes Event Fluorescence Funding G-Quartets Genome Goals Growth Holoenzymes Human Hydroxyl Radical In Vitro Individual Laboratories Length Malignant Neoplasms Maps Modeling Movement Mutation N-terminal Nucleic Acids Nucleoproteins Organism Pharmaceutical Preparations Premature aging syndrome Process Property Protein Subunits Proteins RNA RNA-Directed DNA Polymerase Regulation Research Resolution Ribonucleoproteins Role Series Site Structural Models Structure Techniques Telomerase Telomerase RNA Component Tertiary Protein Structure Testing Tetrahymena Tetrahymena thermophila Therapeutic Work biophysical techniques cell type combat enzyme model in vivo loss of function mutant novel novel strategies programs public health relevance recombinational repair reconstitution research study response single molecule telomere

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 processive extension of telomere DNA using a unique catalytic mechanism that requires a strong functional interdependence of the telomerase RNA, telomerase reverse transcriptase, and several additional protein subunits. The primary objective of this proposal is to elucidate how conserved structural RNA and protein domains coordinate the processes of telomerase RNP assembly, catalysis, and regulation. To address the substantial challenges associated with structural analysis of telomerase we will utilize a multi-faceted experimental strategy that combines single-molecule biophysical techniques paired with computational, biochemical, and high-resolution structural approaches. We will study human telomerase and the enzyme from the model system Tetrahymena thermophila. In aim 1, we will employ x-ray crystallography, single-molecule Förster resonance energy transfer (smFRET), and biochemical probing to characterize local and global structural features of the telomerase complex. Distance constraints furnished by these experiments will aid telomerase structural modeling in collaboration with Nikolai Ulyanov (UCSF). In aim 2, we will use smFRET and small angle x-ray scattering (SAXS) to analyze the role(s) of telomere DNA structure and telomerase-associated proteins (POT1-TPP1, human; Teb1, Tetrahymena) in the regulation of telomerase-DNA interactions and catalysis. This work will be conducted in collaboration with Kathleen Collins (UCB) and Greg Hura (LBNL). In aim 3, we will exploit several novel single-molecule assays to critically evaluate existing models for telomerase conformational dynamics during processive telomere DNA synthesis.

描述（由适用提供）：维持端粒需要的端粒酶核糖核蛋白（RNP），这是保护真核染色体的专门核蛋白结构，这些结构终止于异常的加工和有害的端到端融合事件。端粒酶使用独特的催化机制催化端粒DNA的处理器扩展，该机制需要强大的功能相互依存关系，端粒酶RNA，端粒酶逆转录酶和几个其他蛋白质亚基。该提案的主要目的是阐明构成结构RNA和蛋白质结构域如何协调端粒酶RNP组装，催化和调节的过程。为了解决与端粒酶结构分析相关的重大挑战，我们将利用一种多方面的实验策略，该策略结合了单分子生物物理技术，与计算，生化和高分辨率结构方法配对。我们将研究人类端粒酶和模型系统四膜菌的酶。在AIM 1中，我们将采用X射线晶体学，单分子Förster共振能量转移（SMFRET）和生化探测来表征端粒酶复合物的局部和全局结构特征。这些实验提供的距离约束将与尼古拉·乌利亚诺夫（UCSF）合作有助于端粒酶结构建模。在AIM 2中，我们将使用SMFRET和小角度X射线散射（SAX）来分析端粒DNA结构和与端粒酶相关的蛋白（POT1-TPP1，Human; Teb1，Tetrahymena）的作用。这项工作将与Kathleen Collins（UCB）和Greg Hura（LBNL）合作进行。在AIM 3中，我们将利用几种新型的单分子测定法，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。