Coupling of structure and dynamics in RNA catalysis

RNA催化中结构与动力学的耦合

基本信息

批准号：
8024193
负责人：
Barbara Lynn Golden
金额：
$ 32.77万
依托单位：
PURDUE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-01-01 至 2015-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8024193
关键词：
Acids Active Sites Age Biochemical Biochemistry Biological Assay Biology Catalysis Catalytic RNA Charge Cleaved cell Computer Simulation Coupling Crystallography Cytosine Development Devices Disease Distal Enzymes Functional RNA Gene Expression Gene Expression Profile Gene Expression Regulation Genomics Goals Hepatitis Delta Virus Human Ions Kinetics Knowledge Learning Left Ligands Maps Mechanics Metal Ion Binding Metals Modeling Molecular Molecular Conformation Molecular Models Motion Nature Nucleotides Orphan Outcome Oxygen Pathway interactions Play Positioning Attribute RNA RNA Sequences Raman Spectrum Analysis Reaction Regulation Resolution Role Series Site Solutions Structure System Testing Work X-Ray Crystallography analog base chemical reaction cofactor design divalent metal foot inorganic phosphate insight interest knowledge base magnesium ion molecular dynamics molecular modeling nucleobase quantum research study therapeutic target three dimensional structure

项目摘要

DESCRIPTION (provided by applicant): The broad goals of this proposal are to provide a molecular-level understanding of how RNA enzymes (ribozymes) catalyze chemical reactions. We are studying a self-cleaving RNA that was originally identified in the human hepatitis delta virus, but is now known to be widely distributed in nature. This ribozyme harnesses a nucleobase with a dramatically shifted pKA and a divalent metal ion to catalyze an RNA cleavage reaction. We will integrate X-ray crystallography, molecular dynamics, and solution biochemistry experiments to learn how the three dimensional structure of the RNA interacts with its metal cofactors to achieve catalysis and how the molecular motions of this dynamic RNA contribute to its reactivity. Our first specific aim describes the strategies we will use to solve the three dimensional structure using X-ray crystallography, and to verify that the conformation and any disorder observed in the crystal mimics the conformation of the active ribozyme in solution. In our second specific aim, we will use molecular dynamics to characterize the motions that occur within the ribozyme active site and to understand the role of disorder in ribozyme catalysis. The last specific aim describes biochemical and spectroscopic experiments designed to dissect the contributions of active site components to catalysis. We will analyze potential ligands to the catalytic metal ion, probe the mechanism by which the catalytic metal ion contributes to catalysis, and explore using solution biochemistry the positioning and motions of nucleotides upstream of the scissile phosphate and how they contribute to the reaction pathway. The results of this study will provide an in-depth structural and mechanistic analysis of one ribozyme. However, in the post genomic age, we are seeing an unexpected contribution of non-coding RNA sequences to regulation of gene expression. Ribozymes and riboswitches are being discovered in a variety of contexts, including within eukaryotic transcriptomes. It is therefore essential to have in our knowledge base some in-depth knowledge of a few paradigm systems such as the hepatitis delta virus ribozyme in order to fully understand the catalytic potential of common ribozymes and unique orphan ribozymes. PUBLIC HEALTH RELEVANCE: In the post genomic age, we are seeing an unexpected contribution of non-coding RNA sequences, including ribozymes, to regulation of prokaryotic and eukaryotic gene expression. To fully understand how these non-coding, functional RNAs work, we are undertaking an in depth structural and mechanistic analysis of a ribozyme. We anticipate that the results of this study will provide clues as to the catalytic strategies of many ribozymes, some of which will be therapeutic targets.

描述（由申请人提供）：该提案的总体目标是提供对 RNA 酶（核酶）如何催化化学反应的分子水平理解。我们正在研究一种自切割RNA，这种RNA最初是在人类丁型肝炎病毒中发现的，但现在已知它在自然界中广泛分布。这种核酶利用 pKA 发生显着变化的核碱基和二价金属离子来催化 RNA 裂解反应。我们将结合X射线晶体学、分子动力学和溶液生物化学实验来了解RNA的三维结构如何与其金属辅因子相互作用以实现催化作用以及这种动态RNA的分子运动如何有助于其反应性。我们的第一个具体目标描述了我们将使用的策略来使用 X 射线晶体学解析三维结构，并验证晶体中观察到的构象和任何无序模仿溶液中活性核酶的构象。在我们的第二个具体目标中，我们将利用分子动力学来表征核酶活性位点内发生的运动，并了解核酶催化中无序的作用。最后一个具体目标描述了旨在剖析活性位点组分对催化的贡献的生化和光谱实验。我们将分析催化金属离子的潜在配体，探讨催化金属离子促进催化的机制，并利用溶液生物化学探索可裂变磷酸上游核苷酸的定位和运动以及它们如何对反应途径做出贡献。这项研究的结果将对一种核酶进行深入的结构和机制分析。然而，在后基因组时代，我们看到非编码 RNA 序列对基因表达调控做出了意想不到的贡献。核酶和核糖开关在多种环境中被发现，包括在真核转录组中。因此，我们的知识库中有必要对一些范式系统（例如丁型肝炎病毒核酶）有深入的了解，以便充分了解常见核酶和独特的孤儿核酶的催化潜力。公共卫生相关性：在后基因组时代，我们看到非编码 RNA 序列（包括核酶）对原核和真核基因表达的调节做出了意想不到的贡献。为了充分了解这些非编码功能性 RNA 的工作原理，我们正在对核酶进行深入的结构和机制分析。我们预计这项研究的结果将为许多核酶的催化策略提供线索，其中一些将成为治疗靶点。