Investigating the Catalytic Mechanism of the HDV Ribozyme

HDV 核酶催化机制的研究

• 批准号: 8465171
• 负责人: Joseph Anthony Piccirilli
• 金额: $ 35.29万
• 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/8465171
• 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.

描述（由申请人提供）：定义HDV核酶的催化机理。肝炎Delta病毒（HDV）是一种人类病原体，会引起严重的急性和慢性肝病。病毒基因组包含对复制必不可少的核酶的序列。尽管我们对核酶催化的理解在发现以来的两年半中已经大大提高了，但它们的催化机制仍然在很大程度上不确定。尽管已经解决了许多这些核酶的晶体结构，但结构数据和功能实验之间存在显着差异，包括HDV核酶在内的许多核酶系统。另外，大多数核酶的过渡状态结构在很大程度上仍未确定。在此提案中，我们将采用强大的化学诱变方法与原子突变循环一起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状态之间的连接。同时，我们将在动力学同位素效应（KIE）分析（与Case Western Reserve University的迈克尔·哈里斯（Michael Harris）合作）中应用新开发的最先进技术，以表征反应途径，并获得衡量催化过程中发生的粘结变化的度量。然后，我们将将原子诱变与KIE测量结合在一起，以确定核酶中的功能相互作用如何影响过渡态的键合。这种方法的这种组合在RNA酶学中是前所未有的，并有望在该RNA的催化机理中产生深入的新见解。总体而言，这项工作将建立一个实验和概念范式，用于研究其他RNA酶和核苷酸转移酶，对生物催化的影响将是深远的。