Investigating the Catalytic Mechanism of the HDV Ribozyme

描述(申请人提供)：定义HDV核酶的催化机制。丁型肝炎病毒(HDV)是一种人类病原体，可导致严重的急性和慢性肝病。病毒基因组包含核酶的序列，核酶是复制所必需的。尽管在核酶发现后的25年里，我们对核酶催化的了解有了很大的进步，但它们的催化机制在很大程度上仍然不清楚。虽然许多核酶的晶体结构已经被解决，但包括HDV核酶在内的许多核酶系统的结构数据和功能实验之间的显著差异仍然存在。此外，大多数核酶的过渡态结构在很大程度上仍未确定。在这个建议中，我们将使用强大的化学诱变方法和原子突变循环来揭示分子中对功能至关重要的原子连接，包括核酶、催化基团和反应过渡态之间的连接。同时，我们将应用最新开发的动态同位素效应(KIE)分析技术(与凯斯西储大学的Michael Harris合作)来表征反应途径，并获得在催化过程中发生的成键变化的测量结果。然后，我们将结合原子突变和Kie测量来确定核酶中的功能相互作用如何影响过渡态的键。这种方法的结合在RNA酶学中是史无前例的，并有望对这种RNA的催化机制产生具有穿透力的新见解。总体而言，这项工作将为研究其他RNA酶和核苷酸转移酶建立一个实验和概念范例，对生物催化的影响将是深远的。 公共卫生相关性：HDV是一种人类病原体，会导致人类严重的急性和慢性肝病。病毒基因组包含病毒复制所需的核酶(HDV Ribozyme)，人类基因组包含类似的未知功能序列。了解核酶的作用机制有望揭示RNA催化的基本方面，并揭示病毒致病性的关键特征，这可能会导致新的肝炎治疗方法。

DESCRIPTION (provided by applicant): Defining the Catalytic Mechanism of the HDV Ribozyme. The hepatitis delta virus (HDV) is a human pathogen that causes severe acute and chronic liver disease. The viral genome contains the sequence for a ribozyme that is essential for replication. Although our understanding of ribozyme catalysis has advanced enormously in the two and half decades since their discovery, their mechanisms of catalysis remain largely undefined. Although crystal structures for many of these ribozymes have been solved, significant discrepancies between the structural data and functional experiments remain with many ribozyme systems including the HDV ribozyme. Additionally transition state structure for most ribozymes remains largely undetermined. In this proposal we will employ powerful chemical mutagenesis approaches together with atomic mutation cycles to reveal atomic connections in the molecule that are critical for function, including the connections between the ribozyme, the catalytic groups, and the reaction transition state. Concurrently, we will apply newly developed state of the art techniques in kinetic isotope effect (KIE) analysis (in collaboration with Michael Harris at Case Western Reserve University) to characterize the reaction pathway and obtain a measure of the bonding changes that occur during catalysis. We will then combine atomic mutagenesis with KIE measurements to determine how functional interactions in the ribozyme influence bonding in the transition state. This combination of approaches is unprecedented in RNA enzymology and promises to yield penetrating new insights into the catalytic mechanism of this RNA. Overall this work will establish an experimental and conceptual paradigm for studying other RNA enzymes and nucleotidyl transferases and the implications for biological catalysis will be far-reaching. PUBLIC HEALTH RELEVANCE: HDV is a human pathogen causing severe acute and chronic liver disease in humans. The viral genome contains a ribozyme (HDV ribozyme) required for viral replication, and the human genome contains a similar sequence of unknown function. Understanding the ribozyme's mechanism of action promises to shed light on fundamental aspects of RNA catalysis and reveal critical features of viral pathogenicity, which may lead to new treatments for hepatitis.