Investigating the Catalytic Mechanism of the HDV Ribozyme

HDV 核酶催化机制的研究

• 批准号: 8277979
• 负责人: Joseph Anthony Piccirilli
• 金额: $ 37.55万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-06-01 至 2015-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8277979
• 关键词: Acids; Active Sites; Acute; Adopted; Biological; Catalysis; Catalytic RNA; Chemicals; Collaborations; Complement; Conflict (Psychology); Data; Enzymatic Biochemistry; Enzymes; Genome; Hepatitis; Hepatitis Delta Virus; Human; Human Genome; Hydroxyl Radical; Ions; Isotopes; Kinetics; Laboratories; Lead; Left; Length; Light; Location; Maps; Measurement; Measures; Mediating; Metals; Modeling; Modification; Mutagenesis; Mutation; Nucleotides; Oxygen; Pathogenicity; Pathway interactions; RNA; Reaction; Rest; Site; Structure; System; Techniques; Transferase; Universities; Viral; Viral Genome; Virus Replication; Work; biological systems; chronic liver disease; enzyme structure; functional group; infancy; insight; mutant; novel strategies; nucleobase; nucleotide analog; pathogen; public health relevance; research study

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.

描述（由申请人提供）：定义 HDV 核酶的催化机制。丁型肝炎病毒（HDV）是一种导致严重急性和慢性肝病的人类病原体。病毒基因组包含复制所必需的核酶序列。尽管自核酶被发现以来的二十五年里，我们对核酶催化的理解取得了巨大进步，但它们的催化机制在很大程度上仍不清楚。尽管许多核酶的晶体结构已得到解决，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仍然存在显着差异。此外，大多数核酶的过渡态结构在很大程度上仍未确定。在本提案中，我们将采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，我们将应用新开发的最先进的动力学同位素效应（KIE）分析技术（与凯斯西储大学的迈克尔哈里斯合作）来表征反应途径并获得催化过程中发生的键合变化的测量。然后，我们将原子诱变与 KIE 测量相结合，以确定核酶中的功能相互作用如何影响过渡态的键合。这种方法的结合在 RNA 酶学领域是前所未有的，有望为这种 RNA 的催化机制带来深入的新见解。总的来说，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式，并且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，可导致人类严重的急性和慢性肝病。病毒基因组含有病毒复制所需的核酶（HDV核酶），人类基因组含有类似但功能未知的序列。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，这可能会带来肝炎的新治疗方法。