Coupling of structure and dynamics in RNA catalysis

RNA催化中结构与动力学的耦合

• 批准号: 8206551
• 负责人: Barbara Lynn Golden
• 金额: $ 30.19万
• 依托单位: PURDUE UNIVERSITY
• 依托单位国家: 美国
• 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/8206551
• 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; public health relevance; quantum; research study; therapeutic target; three dimensional structure

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）：该提案的主要目标是提供对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我切割的RNA，这种RNA最初在人类丁型肝炎病毒中被发现，但现在已知它在自然界中广泛分布。这种核酶利用具有显著移位的pKA和二价金属离子的核碱基来催化RNA切割反应。我们将整合X射线晶体学，分子动力学和溶液生物化学实验，以了解RNA的三维结构如何与其金属辅因子相互作用以实现催化，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X射线晶体学来解决三维结构的策略，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，我们将使用分子动力学来表征核酶活性位点内发生的运动，并了解核酶催化中的无序作用。最后一个具体的目标描述了生物化学和光谱实验，旨在解剖活性部位组分的催化作用的贡献。我们将分析潜在的催化金属离子的配体，探测催化金属离子有助于催化的机制，并利用溶液生物化学探索易裂磷酸盐上游核苷酸的定位和运动，以及它们如何有助于反应途径。本研究的结果将提供一个深入的结构和机制分析的核酶。然而，在后基因组时代，我们看到了非编码RNA序列对基因表达调控的意想不到的贡献。核酶和核糖开关被发现在各种情况下，包括在真核转录组。因此，在我们的知识库中有一些深入的知识，如丁型肝炎病毒核酶的一些范例系统，以充分了解常见的核酶和独特的孤儿核酶的催化潜力是至关重要的。 公共卫生相关性：在后基因组时代，我们看到了非编码RNA序列，包括核酶，对原核和真核基因表达的调控的意想不到的贡献。为了充分了解这些非编码的功能性RNA是如何工作的，我们正在对核酶进行深入的结构和机制分析。我们预计，这项研究的结果将提供线索，许多核酶的催化策略，其中一些将是治疗目标。