Hairpin Ribozyme: Folding, Structure and Mechanism

描述（由申请人提供）提出了一系列生物化学、生物物理学、组合和分子模拟实验，旨在解决关于发夹状核酶的三个基本问题。首先，RNA如何折叠成其天然结构？第二，主动站点架构的关键特征是什么？第三，催化机制是什么？最近的进展使在下一个资助期回答这些问题成为可能。这些进展包括：（a）开发在分子和核碱基水平上分析折叠事件的方法，（B）证明实验室生物化学和计算方法的组合可以导致活性位点处的特定三级相互作用的建模和实验验证，（c）阐明核酶的晶体结构，（d）发现G8参与非金属依赖性活性位点化学，以及（e）通过体外选择执行复杂“遗传”分析的能力。具体目标是：（1）确定催化和折叠中涉及的特定核苷酸和官能团的结构和功能作用;（2）通过体外选择分离失活突变的拟回复突变体，并建立功能补偿机制;（3）表征和模拟催化循环过程中的构象变化，并确定哪些是催化功能所必需的;（4）开发和测试催化机制的模型。这项工作的生物医学重要性包括：（i）我们对RNA结构及其如何导致催化活性的理解的进步，（ii）理解由核酶和核糖核蛋白复合物催化的生物反应（包括核糖体和剪接体）在生物反应中的应用，以及（iii）我们对催化RNA和核酶工程的理解在改善健康方面的应用，通过功能基因组学，药物靶标验证，以及基于选择性核酶的遗传和病毒性疾病治疗剂的开发。

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.