NMR and EPR Studies of Ribozyme Catalytic Mechanisms

核酶催化机制的 NMR 和 EPR 研究

基本信息

批准号：
7931125
负责人：
CHARLES G HOOGSTRATEN
金额：
$ 7万
依托单位：
MICHIGAN STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-09-30 至 2010-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7931125
关键词：
Address Affect Binding Binding Sites Biochemical Energetics Biological Assay Catalysis Catalytic Domain Catalytic RNA Chemistry Complement Complex Coupled Coupling DNA Sequence Rearrangement Data Development Disease Docking Goals Heteronuclear NMR Ions Kinetics Label Left Ligation Link Magnetic Resonance Maps Messenger RNA Metal Ion Binding Metals Methods Molecular Molecular Conformation Molecular Structure Mutation Pathway interactions Physiologic pulse Play Positioning Attribute Process Property RNA RNA Splicing Reaction Relaxation Research Personnel Role Scheme Site Spliceosomes Structure Substrate Domain System Techniques Testing Therapeutic Thermodynamics U6 small nuclear RNA Ursidae Family Variant Work catalyst cofactor comparative design divalent metal fascinate hairpin ribozyme interest leadzyme mRNA Precursor new technology novel programs research study stem tool

项目摘要

DESCRIPTION (provided by applicant): The overarching goal of this project is to develop a detailed biophysical understanding of the mechanism of action of catalytic RNA molecules, or ribozymes. Ribozyme derivatives are currently under development as therapeutics for a number of devastating diseases. A fundamental understanding of ribozyme mechanism is critical in the design of derivatives, with necessary improvements in pharmacological properties that simultaneously maintain the desired catalytic activity. Our thesis, is that, the static structure of a ribozyme is insufficient to understand its mechanism of action, and that structural work must be coupled with studies of the molecule's dynamics, its interactions with multivalent metal ions, and the effects of those metal ions on molecular structure and dynamics. We will apply contemporary magnetic resonance techniques, including heteronuclear NMR spin relaxation and advanced EPR experiments, to provide unique information that bears directly on these issues and, thus, to link structural studies with biochemical energetics. The dynamic properties of ribozyme conformations, as studied by NMR spin relaxation, will be emphasized. In the well characterized hairpin ribozyme, we will investigate the dynamic properties of the molecule using NMR spin relaxation in active vs. inactive sequence variants, and as a function of tertiary structure formation (docking). We will proceed to use an integrated EPR and NMR approach, to delineate the effects of multivalent metal ions, which are crucial for catalysis, but do not participate directly in chemistry in this system, on the ribozyme's structure and dynamics. We are also interested in the U6 snRNA, a catalytically critical component of the eukaryotic mRNA splicing apparatus. In this system, we will investigate the structure of a biochemically-identified metal ion, which appears to participate in reaction chemistry, both in the internal stemloop that forms its immediate binding site, and in larger complexes, including pre-mRNA sequences. Studies of these two systems will provide complementary and synergistic perspectives on the interrelationships among conformational dynamics, metal ion cofactor ligation, and catalytic function in RNA. In short, we propose an integrated biophysical program of technological progress and novel applications, that will significantly advance the ribozyme field toward a molecular-level understanding of these fascinating catalysts.

描述（由申请人提供）：该项目的总体目标是对催化RNA分子或核酶作用机理的详细生物物理理解。核酶衍生物目前正在开发作为多种毁灭性疾病的治疗剂。对核酶机制的基本理解在衍生物的设计中至关重要，在同时维持所需的催化活性的药理特性的必要改善中。我们的论点是，核酶的静态结构不足以理解其作用机理，并且必须将结构工作与分子动力学的研究，与多价金属离子的相互作用以及这些金属离子对分子结构和动力学的影响。我们将采用当代磁共振技术，包括异核NMR自旋松弛和高级EPR实验，以提供直接在这些问题上的独特信息，从而将结构研究与生化能量学联系起来。 NMR自旋弛豫研究的核酶构象的动态特性将得到强调。在表征良好的发夹核酶中，我们将使用活性与非活动序列变体中的NMR自旋弛豫研究分子的动态特性，并且是第三级结构形成（docking）的函数。我们将继续使用集成的EPR和NMR方法来描述多价金属离子的影响，这些金属离子对于催化至关重要，但不直接参与该系统中的化学，对核酶的结构和动力学。我们也对U6 snRNA感兴趣，U6 snRNA是真核mRNA剪接设备的催化关键成分。在该系统中，我们将研究生化识别的金属离子的结构，该金属离子似乎参与了反应化学，既在形成其直接结合位点的内部茎卢比中，又在包括前MRNA序列（包括前MRNA序列）中。对这两个系统的研究将提供有关RNA中构象动力学，金属离子辅因子结扎和催化功能之间相互关系的互补观点。简而言之，我们提出了一个综合的技术进步和新应用生物物理计划，该计划将大大推进核酶领域的分子水平对这些迷人的催化剂的理解。