Coupling of structure and dynamics in RNA catalysis

描述（由申请人提供）：本提案的总体目标是提供RNA酶（核酶）如何催化化学反应的分子水平的理解。我们正在研究一种自我切割的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。这种核酶利用一个具有显著移位pKA的核碱基和一个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，以及这种动态RNA的分子运动如何促进其反应性。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，并验证在晶体中观察到的构象和任何无序都模仿了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，并了解核酶催化中紊乱的作用。最后一个具体目的是描述生化和光谱实验，旨在剖析活性位点成分对催化的贡献。我们将分析催化金属离子的潜在配体，探索催化金属离子参与催化的机制，并利用溶液生物化学方法探索可裂磷酸盐上游核苷酸的定位和运动及其对反应途径的贡献。本研究的结果将为一种核酶提供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的意想不到的贡献。核酶和核开关在各种情况下被发现，包括在真核生物转录组中。因此，有必要在我们的知识库中深入了解一些范例系统，如丁型肝炎病毒核酶，以便充分了解普通核酶和独特的孤儿核酶的催化潜力。

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.