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Mechanistic Analysis of an RNA Enzyme In Vivo

体内 RNA 酶的机理分析

基本信息

批准号：
7253347
负责人：
Martha J. Fedor
金额：
$ 33.79万
依托单位：
SCRIPPS RESEARCH INSTITUTE, THE
依托单位国家：
美国
项目类别：
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/7253347
关键词：
Adopted Award Bacterial Gene Expression Regulation Base Pairing Behavior Binding Sites Biological Biological Models Biological Process Catalytic RNA Cells Cleaved cell Code Complex DNA Sequence Rearrangement Development Environment Enzymes Equilibrium Event Functional RNA Genetic Genetic Transcription Glean Goals Hand In Vitro Individual Ions Kinetics Learning Life Ligand Binding Ligands Ligation Maintenance Measures Mediating Messenger RNA Modeling Molecular Molecular Conformation Monitor Numbers Pathway interactions Play Post-Translational Protein Processing Property Proteins RNA RNA Folding RNA Probes RNA Processing RNA analysis RNA-Binding Proteins Reaction Reagent Reporting Research Research Design Research Personnel Ribosomes Role Site-Directed Mutagenesis Small Nucleolar RNA Small RNA Specificity Structure Structure-Activity Relationship System Testing Therapeutic Therapeutic Intervention Thermodynamics Transcription Process Translating Translations Tube Untranslated Regions Variant Viral Yeasts base conformational conversion design hairpin ribozyme in vivo insight messenger ribonucleoprotein programs rRNA Precursor research study small molecule therapeutic target viral RNA

项目摘要

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分，为治疗干预提供潜在的治疗靶标和试剂。RNA组装反应和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介导的生物过程中，特定RNA折叠事件的定量分析由于许多组分参与复杂途径而变得复杂。另一方面，RNA酶直接通过其催化活性报告功能性RNA结构的组装。使用RNA酶作为体外模型系统的RNA折叠研究已经产生了大量关于RNA介导的反应的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细胞复杂环境中RNA的行为。发夹状核酶是体内RNA结构-功能研究的特别有价值的模型，因为核酶变体已经被设计成使得细胞内切割活性监测沿着反应途径的特定RNA构象转变沿着。具体目标是1）使用分子间核酶切割活性来探测体内RNA复合物形成的特异性、动力学和平衡，2）使用具有采用确定的非功能性二级结构的潜力的核酶变体来区分细胞内RNA组装的快速构象交换和延迟折叠模型，3）确定翻译后RNA重折叠途径如何不同于共转录折叠，以及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告特定生物学背景下的RNA结构和动力学的机制见解将有助于阐明更复杂的RNA介导的反应机制，并为基于RNA的治疗方法的开发提供合理的框架。