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：
10528468
负责人：
Michael D Stone
金额：
$ 35.69万
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 2024-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10528468
关键词：
Active Sites Address Adopted Architecture Binding Biochemical Biochemistry Biological Assay Biological Models Biophysics Catalysis Catalytic Domain Cells Characteristics Chromosomes Complex Coupled Cryoelectron Microscopy DNA DNA biosynthesis Data Defect Deterioration Diagnosis Disease Drug Targeting Enzymes Event Fluorescence Resonance Energy Transfer G-Quartets Genetic Recombination Genome Genome Stability Goals Growth Human Kinetics Label Malignant Neoplasms Mediating Methods Molecular Molecular Conformation Movement Mutation N-terminal Nucleoproteins Nucleotides Pathway interactions Positioning Attribute Premature aging syndrome Process Property Protein Dynamics Protein Subunits Proteins RNA RNA-Directed DNA Polymerase Regulation Reporting Research Resolution Reverse Transcription Ribonucleoproteins Site Structure Techniques Telomerase Telomerase RNA Component Telomere Shortening Tertiary Protein Structure Testing Tetrahymena Tetrahymena thermophila Therapeutic Tissues X-Ray Crystallography biophysical techniques cell growth cofactor design detection method enzyme model experimental study flexibility loss of function novel novel strategies polypeptide prevent protein protein interaction recruit repaired scaffold single molecule telomere

项目摘要

Project Summary 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 fu- sion events. Telomerase catalyzes processive extension of telomere DNA via a unique 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 RNA and protein domains coordinate the processes of telomerase RNP assembly, cataly- sis, and recruitment to telomeres. To address this goal, 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 biochemical structure probing, single-molecule Förster resonance energy transfer (smFRET), and x-ray crystallography to analyze structural intermediate states adopted by telomerase RNA and TERT during the RNP assembly pathway. In aim 2, we will use smFRET and a novel telomerase ac- tivity detection method to investigate conformational dynamics of protein and RNA domains that drive telomer- ase function. In aim 3, we will study the dynamic DNA handling properties of the telomerase enzyme. These experiments will focus on the molecular mechanisms for telomerase recruitment to telomeres as well as under- standing how the intrinsic folding properties of telomere DNA regulate telomerase catalysis.

项目概要端粒酶核糖核蛋白 (RNP) 是维持端粒（一种特殊的核蛋白）所必需的保护真核染色体末端免受异常加工和有害的端到端功能的结构端粒酶通过独特的催化机制催化端粒 DNA 的持续延伸。这需要端粒酶RNA、端粒酶逆转录酶在功能上有很强的相互依赖性（TERT）和几个额外的蛋白质亚基该提案的主要目的是阐明如何。保守的结构RNA和蛋白质结构域协调端粒酶RNP组装、催化的过程为了实现这一目标，我们将利用多方面的实验策略将单分子生物物理技术与计算、生物化学和高分辨率相结合我们将研究人类端粒酶和来自模型系统四膜虫的酶。在目标 1 中，我们将采用生化结构探测、单分子福斯特共振能量。转移 (smFRET) 和 X 射线晶体学分析端粒酶采用的结构中间态 RNP 组装途径中的 RNA 和 TERT 在目标 2 中，我们将使用 smFRET 和一种新型端粒酶 ac-。活性检测方法研究驱动端粒的蛋白质和RNA结构域的构象动力学在目标 3 中，我们将研究端粒酶的动态 DNA 处理特性。实验将集中于端粒酶招募至端粒的分子机制以及端粒酶的分子机制。端粒 DNA 的内在折叠特性如何调节端粒酶催化。