Catalytic Mechanisms of RNA Ezymes

RNA酶的催化机制

基本信息

批准号：
8008948
负责人：
Martha J. Fedor
金额：
$ 10.77万
依托单位：
SCRIPPS RESEARCH INSTITUTE, THE
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：
2010-01-28 至 2010-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8008948
关键词：
5&apos Untranslated Regions 8-azaadenosine 8-azapurine Acids Active Sites Address Amines Architecture Award Binding Biochemical Biological Assay Biology Catalysis Catalytic RNA Cations Chemicals Chemistry Cleaved cell Complex Dependence Development Disease Dissociation Enzymes Equation Equilibrium Event Fluorescence Free Energy Functional RNA Gene Expression Regulation Goals Growth and Development function Health Hepatitis Delta Virus Heteronuclear NMR Hydrogen Bonding Individual Introns Kinetics Left Ligands Measures Mediating Metabolic Metals Methods Microscopic Modeling Molecular Monitor Mutagenesis Oxygen Pathway interactions Peptidyltransferase Protein Biosynthesis Proteins Protons RNA RNA Folding RNA Splicing Reaction Reagent Research Personnel Resolution Ribosomes Role Structure Sulfur Testing Therapeutic Thermodynamics Work azaguanosine base catalyst cofactor deprotonation functional group hairpin ribozyme inorganic phosphate insight nucleobase phosphodiester programs protonation prototype research study role model

项目摘要

Catalytic RNAs provide a window into a primordial 'RNA World' from which modern biology might have evolved. Contemporary roles for RNA catalysts in the modern protein world are still found in the regulation of gene expression and in protein synthesis, making RNA catalysis essential to normal growth and development. Recent high-resolution structures of self-cleaving and self-splicing RNAs provide pictures of the active sites for the hammerhead, hepatitis delta virus, and hairpin self-cleaving RNAs and Group-l self- splicing introns. The structure of the ribosome gave us a view of a peptidyl transferase center constructed entirely from RNA. Mechanistic studies carried out over more than 20 years since ribozymes were first discovered can now be compared and contrasted with these structures, laying the groundwork for experiments to probe fundamental questions about how RNA enzymes use their functional groups for catalysis. As the first ribozyme shown to function without metal cation cofactors, the hairpin ribozyme is a prototype for understanding metal-independent RNA catalysis. Work during previous award periods defined the essential structural and biochemical features of the active site and led to testable models for the roles of active site nucleobases in catalytic chemistry. Our goal for the coming award period is to define the catalytic mechanism of the hairpin ribozyme and elucidate the mechanisms of a recently discovered metabolite- dependent ribozyme, which resembles the hairpin ribozyme in certain key mechanistic features. Specific goals include 1) distinguishing contributions to bond breaking and bond making steps in the hairpin ribozyme reaction using 5' phosphorothiolate RNAs, 2) determining the microscopic pKa values of hairpin ribozyme active site nucleobases using 8-azapurine fluorescence to monitor pH-dependent changes in protonation states and using heteronuclear NMR to monitor pH-dependent changes in chemical shifts, 3) determine the energetic contributions of individual active site functional groups to ground state and transition state stability by combining atomic level mutagenesis with physical and functional assays of hairpin ribozyme complex formation and catalysis, and 4) establish a kinetic and thermodynamic framework for quantitative analysis of the reaction pathway of the metabolite-responsive glmS ribozyme. The proposed studies will generate fundamental insights into mechanisms of catalysis by RNA and RNP enzymes, RNA folding and dynamics, and RNA interactions with small ligands. This work will deepen our basic understanding of RNA structure and function, which is a critical part of the fundamental biology of health and disease. The results of these studies also will provide a framework to guide the development of technical and therapeutic applications involving RNAs as targets and reagents.

催化RNA为现代生物学可能从中提供了一个原始的“ RNA世界”的窗口已经进化了。现代蛋白质世界中RNA催化剂的当代角色仍在调节基因表达和蛋白质合成中的调节，使RNA催化对正常生长至关重要发展。最近的自我裂解和自剪接RNA的高分辨率结构提供了图片锤头，乙型肝炎病毒和发夹自我切换的RNA和l组自我自我的主动部位剪接内含子。核糖体的结构为我们提供了构造的肽基转移酶中心的视图完全来自RNA。自核酶首次进行了20多年的机械研究现在可以比较发现并与这些结构进行对比，为实验以探测有关RNA酶如何使用其功能组的基本问题催化。由于第一个核酶显示在没有金属阳离子辅因子的情况下起作用，因此发夹核酶为一个理解非金属依赖性RNA催化的原型。在定义的以前的奖励期间工作活性位点的基本结构和生化特征，并导致了可测试的模型催化化学中的活性位点核酶。我们即将到来的奖项时期的目标是定义催化发夹核酶的机制，并阐明了最近发现的代谢物的机制依赖性核酶，类似于某些关键机械特征的发夹核酶。具体的目标包括1）区分发夹核酶中债券断裂和债券的贡献使用5'磷酸酯RNA的反应，2）确定发夹核酶的微观PKA值活性位点核碱基使用8-氮杂氨酸荧光监测质子化的pH依赖性变化状态并使用异核NMR监测化学位移的pH依赖性变化，3）确定单个主动部位官能团对基态和过渡状态稳定性的能量贡献通过将原子水平诱变与发夹核酶复合物的物理和功能测定形成和催化，以及4）建立一个动力学和热力学框架，用于定量分析代谢产物反应性GLMS核酶的反应途径。提出的研究将产生 RNA和RNP酶，RNA折叠和动力学的催化机制的基本见解，与小配体的RNA相互作用。这项工作将加深我们对RNA结构的基本理解和功能，这是健康和疾病基本生物学的关键部分。这些结果研究还将提供一个框架来指导技术和治疗应用的开发涉及RNA作为目标和试剂。