MECHANISTIC ANALYSIS OF AN RNA ENZYME IN VIVO

体内 RNA 酶的机理分析

• 批准号: 6228427
• 负责人: Martha J. Fedor
• 金额: $ 29.79万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-03-01 至 2005-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6228427
• 关键词: RNA binding protein; active sites; allosteric site; chemical structure function; conformation; enzyme mechanism; messenger RNA; nucleic acid metabolism; nucleic acid structure; ribozymes; synthetic enzyme

DESCRIPTION (adapted from applicant's abstract): The goal of the proposed research is to exploit the hairpin ribozyme and the power of mechanistic enzymology to develop a detailed, quantitative view of the kinetics and equilibria of RNA-mediated reactions in living cells. RNAs are critical components of the biological machinery responsible for maintenance and expression of genetic information, providing potential targets and reagents for therapeutic intervention. The dynamics and equilibria of RNA conformational transitions play key roles in transcription, translation and RNA processing reactions. Determining the functional significance of specific structures and conformational transitions in RNA-mediated biological processes is complicated by the participation of many components in complex pathways. On the other hand, mechanistic studies of RNA enzymes in vitro have produced detailed, quantitative descriptions of the kinetics and equilibria of individual steps of RNA-mediated reactions and the influence of small molecules, RNA binding proteins, and RNA transcription on RNA assembly pathways. The hairpin ribozyme provides an especially valuable model system for RNA structure-function studies in vivo because ribozyme variants can be designed so that intracellular cleavage activity monitors specific RNA conformational transitions along the reaction pathway. Mechanistic insights gleaned using this simple system to report on RNA structure and dynamics in specific biological contexts will help illuminate mechanisms of more complex RNA-mediated reactions and provide a rational framework for the development of RNA-based therapeutics. Specific goals include: establishing a kinetic and thermodynamic framework for mechanistic studies of RNA conformational transitions in vivo, determining whether RNA structures display different conformational equilibria and dynamics in nuclear and cytoplasmic compartments, determining how transcription and translation influence RNA assembly pathways in vivo, characterizing allosteric regulation of RNA structure and function by small molecule and protein effectors in vivo, and developing a system for ribozyme-mediated mRNA repair through sequential cleavage and ligation reactions. Mechanistic insights gleaned using the hairpin ribozyme to report on RNA structure and dynamics in specific biological contexts will help illuminate mechanisms of more complex RNA-mediated reactions and provide a rational framework for the development of RNA-based therapeutics.

描述（改编自申请人的摘要）：拟议的目标 研究的目的是开发发夹状核酶和其机制的力量， 酶学发展的详细，定量的动力学观点， 活细胞中RNA介导反应的平衡。RNA至关重要 生物机器的组成部分负责维护和 基因信息的表达，提供潜在的目标和试剂， 治疗干预RNA构象的动力学和平衡 转换在转录、翻译和RNA加工中起关键作用 反应.确定特定结构的功能意义， RNA介导的生物过程中的构象转换是复杂的 是由许多成分参与复杂的途径。另一方面，在一项研究中， 体外RNA酶的机理研究已经产生了详细的， 定量描述的动力学和平衡的个别步骤， RNA介导的反应和小分子的影响，RNA结合 蛋白质和RNA转录的RNA组装途径。 发夹状核酶为研究RNA提供了一个特别有价值的模型系统 因为可以设计核酶变体， 细胞内切割活性监测特定的RNA构象 沿着反应途径转变。机械学的洞察力， 用于报告特定生物中RNA结构和动力学的简单系统 上下文将有助于阐明更复杂的RNA介导的反应机制， 并为基于RNA的疗法的发展提供了合理的框架。 具体目标包括：建立一个动力学和热力学框架， 体内RNA构象转换的机制研究，确定 RNA结构是否表现出不同的构象平衡和动力学 在细胞核和细胞质区室，决定如何转录和 翻译影响体内RNA组装途径，表征变构 小分子和蛋白质对RNA结构和功能的调控 体内效应器，并开发核酶介导的mRNA修复系统 通过连续的切割和连接反应。机械的见解 收集使用发夹核酶报告RNA的结构和动力学， 具体的生物学背景将有助于阐明更复杂的 RNA介导的反应，并提供了一个合理的框架， 基于RNA的治疗方法。