Hairpin Ribozyme: Folding, Structure and Mechanism

发夹核酶：折叠、结构和机制

• 批准号: 6986236
• 负责人: JOHN MacKenzie BURKE
• 金额: $ 49.06万
• 依托单位: UNIVERSITY OF VERMONT & ST AGRIC COLLEGE
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-12-01 至 2009-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6986236
• 关键词: active sites; crosslink; enzyme mechanism; enzyme substrate complex; fluorescence spectrometry; functional /structural genomics; gene mutation; molecular dynamics; nucleic acid structure; protein folding; ribosomes; ribozymes; spliceosomes

DESCRIPTION (provided by applicant) An integrated series of biochemical, biophysical, combinatorial, and molecular simulation experiments is presented, designed to address three fundamental questions concerning the hairpin ribozyme. First, how does the RNA fold into its native structure? Second, what are the key features of active site architecture? Third, what is the catalytic mechanism? Recent advances make it possible to answer these questions during the next funding period. These advances include: (a) development of methods to analyze folding events at the molecular and nucleobase levels, (b) demonstration that a combination of bench biochemistry and computational methods can lead to the modeling and experimental verification of specific tertiary interactions at the active site, (c) elucidation of crystal structures of the ribozyme, (d) discovery that G8 participates in metal-independent active site chemistry, and (e) the ability to execute a sophisticated 'genetic' analysis through in vitro selection. Specific Aims are: (1) Identify the structural and functional roles of specific nucleotides and functional groups implicated in catalysis and folding; (2) Isolate peudorrevertants of inactivating mutations by in vitro selection, and establish the mechanism of functional compensation; (3) Characterize and model conformational changes during the catalytic cycle, and determine which are necessary for catalytic function; (4) Develop and test models of the catalytic mechanism. The biomedical importance of this work includes (i) the advancement of our understanding of RNA structure and how it leads to catalytic activity, (ii) understanding biological reactions catalyzed by ribozymes and ribonucleoprotein complexes (including the ribosome and the spliceosome) in biological reactions, and (iii) the application of our understanding of catalytic RNA and ribozyme engineering to improving health, through functional genomics, pharmaceutical target validation, and the development of selective ribozyme based therapeutics for genetic and viral diseases.

描述（由申请人提供）提出了一系列的生化，生物物理，组合和分子仿真实验的集成系列，旨在解决有关发夹核酶的三个基本问题。首先，RNA如何折叠到其天然结构中？第二，活动站点体系结构的关键功能是什么？第三，什么是催化机制？最近的进步使得在下一个资金期间回答这些问题成为可能。这些进步包括：（a）开发在分子和核碱基水平上分析折叠事件的方法，（b）证明，基准生物化学和计算方法的结合可以导致对活性位点上特定第三级相互作用的建模和实验验证（e）通过体外选择执行复杂的“遗传”分析的能力。具体目的是：（1）确定与催化和折叠有关的特定核苷酸和官能团的结构和功能作用； （2）通过体外选择分离灭活突变的卵形逆想者，并建立功能补偿的机制； （3）在催化循环期间表征和模型构象变化，并确定催化功能的必要条件； （4）开发和测试催化机制。这项工作的生物医学重要性包括（i）我们对RNA结构的理解以及如何导致催化活性的发展，（ii）了解生物学反应是由核酶和核糖核蛋白酶复合物和核糖核蛋白复合物催化的（包括核糖体和酶子体）在生物反应中的生物体和（iii）通过（iii）对猫型型蛋白质的应用，以及（iii）的应用，并通过核酸含量的适用性来提高蛋白质的效果。药物靶标验证，以及针对遗传和病毒疾病的选择性核酶的开发。