Mechanistic Analysis of an RNA Enzyme In Vivo

体内 RNA 酶的机理分析

• 批准号: 7145155
• 负责人: Martha J. Fedor
• 金额: $ 34.8万
• 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 2010-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7145155
• 关键词: RNA binding protein; Saccharomyces cerevisiae; active sites; allosteric site; chemical cleavage; chemical structure function; conformation; enzyme mechanism; fungal genetics; genetic transcription; messenger RNA; molecular assembly /self assembly; nucleic acid metabolism; nucleic acid structure; ribozymes; synthetic enzyme

DESCRIPTION (provided by applicant): The goal of the proposed research is to exploit the simple cleavage and ligation reactions of hairpin ribozymes to assess the dynamics and equilibria of RNA conformational transitions in a biological context. RNAs are critical components of the biological machinery responsible for maintenance and expression of genetic information, providing potential therapeutic targets and reagents for therapeutic intervention. RNA assembly reactions and exchange among alternative RNA structures play key roles in transcription, translation, viral replication and RNA processing reactions. Quantitative analyses of specific RNA folding events in most RNA-mediated biological processes are complicated by the participation of many components in complex pathways. On the other hand, RNA enzymes report on assembly of functional RNA structures directly through their catalytic activity. Studies of RNA folding using RNA enzymes as model systems in vitro have produced a wealth of information regarding 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 in simple test tube reactions. Our goal is to learn how the principles that have been elucidated through in vitro studies translate to the behavior of RNAs in the complex environment of a cell. The hairpin ribozyme is an especially valuable model for RNA structure-function studies in vivo because ribozyme variants have been designed so that intracellular cleavage activity monitors specific RNA conformational transitions along the reaction pathway. Specific goals are 1) to use inter-molecular ribozyme cleavage activity to probe the specificity, kinetics and equilibria of RNA complex formation in vivo, 2) use ribozyme variants with the potential to adopt defined nonfunctional secondary structures to distinguish rapid conformational exchange and delayed folding models of intracellular RNA assembly, 3) determine how post- translational RNA re-folding pathways differ from co-transcriptional folding and 4) characterize the kinetics and equilibria of small ligand-induced RNA conformational transitions in vivo. 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.

描述（由申请人提供）：拟议研究的目标是利用发夹核酶的简单切割和连接反应来评估生物学背景下RNA构象转变的动力学和平衡。rna是负责维持和表达遗传信息的生物机制的关键组成部分，为治疗干预提供了潜在的治疗靶点和试剂。RNA组装反应和不同RNA结构之间的交换在转录、翻译、病毒复制和RNA加工反应中起着关键作用。在大多数RNA介导的生物学过程中，由于复杂途径中许多成分的参与，对特定RNA折叠事件的定量分析变得复杂。另一方面，RNA酶通过其催化活性直接报道功能性RNA结构的组装。利用RNA酶作为模型系统在体外进行的RNA折叠研究，已经产生了大量关于RNA介导反应各个步骤的动力学和平衡，以及小分子、RNA结合蛋白和RNA转录对简单试管反应中RNA组装途径的影响的信息。我们的目标是了解通过体外研究阐明的原理如何转化为rna在细胞复杂环境中的行为。发夹核酶是体内RNA结构-功能研究的一个特别有价值的模型，因为核酶变体已经被设计成细胞内裂解活性监测沿着反应途径的特定RNA构象转变。具体目标是：1)利用分子间核酶裂解活性来探测RNA复合物在体内形成的特异性、动力学和平衡；2)利用核酶变体可能采用已定义的非功能二级结构来区分细胞内RNA组装的快速构象交换和延迟折叠模型。3)确定翻译后RNA重折叠途径与共转录折叠途径的区别；4)表征体内小配体诱导的RNA构象转变的动力学和平衡。使用这个简单的系统来报道特定生物学背景下RNA结构和动力学的机制见解将有助于阐明更复杂的RNA介导反应的机制，并为基于RNA的治疗方法的发展提供合理的框架。