Coupling of structure and dynamics in RNA catalysis

RNA催化中结构与动力学的耦合

• 批准号: 8601108
• 负责人: Barbara Lynn Golden
• 金额: $ 30.14万
• 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/8601108
• 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.

描述（由申请人提供）：该提案的广泛目标是对RNA酶（核酶）如何催化化学反应的分子级别的理解。我们正在研究最初在人类肝炎三角洲病毒中鉴定出的自我切割的RNA，但现在已知在自然界中分布广泛。该核酶可以利用核苷酸酶具有巨大移动的PKA和二价金属离子来催化RNA裂解反应。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助因子相互作用以实现催化以及该动态RNA的分子运动如何有助于其反应性。我们的第一个具体目的描述了我们将使用X射线晶体学来解决三维结构的策略，并验证晶体模拟物中观察到的构象和任何障碍是否在溶液中活性核酶的构象。在我们的第二个特定目标中，我们将使用分子动力学来表征核酶活性位点中发生的运动，并了解疾病在核酶催化中的作用。最后的特定目的描述了旨在剖析活性位点成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，探测催化金属离子有助于催化的机制，并使用溶液生物化学探索磷酸盐磷酸盐上游的核苷酸的定位和运动以及它们如何对反应途径贡献。这项研究的结果将为一个核酶提供深入的结构和机械分析。但是，在基因组后年龄，我们看到非编码RNA序列对基因表达调节的意外贡献。在多种情况下，包括在真核转录组中，正在发现核酶和核糖开关。因此，在我们的知识基础上，必须深入了解一些范式系统，例如肝炎三角洲病毒核酶，以充分了解常见核酶和独特的孤儿核酶的催化潜力。