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

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

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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.

项目摘要维持端粒需要的端粒酶核糖核蛋白（RNP）是必需的保护真核染色体的结构末尾免受异常处理和有害的端到端fu- Sion事件。端粒酶通过独特的催化机制催化端粒DNA的过程扩展这需要端粒酶RNA，端粒酶逆转录酶的强大功能相互依赖性（TERT）和其他几个蛋白质亚基。该提议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远程酶RNP组装的过程，催化 SIS，并招募端粒。为了解决这个目标，我们将利用一种多方面的实验策略结合单分子生物物理技术与计算，生化和高分辨率配对结构方法。我们将研究人类端粒酶和来自模型系统的酶嗜热。在AIM 1中，我们将采用生化结构探测，单分子förster共振能转移（SMFRET）和X射线晶体学以分析端粒酶采用的结构中间状态 RNP组装途径中的RNA和TERT。在AIM 2中，我们将使用SMFRET和新型端粒酶AC- 调查蛋白质和RNA结构域的会议动力学的抗性检测方法 ASE功能。在AIM 3中，我们将研究端粒酶酶的动态DNA处理特性。这些实验将集中在端粒酶募集到端粒以及不足的分子机制上端粒DNA的固有折叠特性如何调节端粒酶催化。