HAIRPIN RIBOZYME--FOLDING, STRUCTURE, AND MECHANISM

一系列综合的生化、生物物理和组合实验旨在回答有关发夹状核酶的三个基本问题：第一，RNA如何组装成催化结构？第二，核酶-底物复合体的结构是什么？第三，这种结构如何导致催化活性的获得？发夹状核酶非常适合于这样的实验，因为它的分子足够小，可以进行全面的研究，但又足够复杂，以至于在催化循环中会发生显著的构象变化。发夹在几个重要方面不同于仅有的两种被更广泛研究的RNA酶，锤头状核酶和第一组内含子。具体目标是：(1)绘制核酶-底物复合体的三级结构图；(2)确定金属离子和抑制剂调节催化活性的机制；(3)定义折叠途径并确定折叠和去折叠的要求、动力学和热力学；(4)以高分辨率确定核酶-底物复合体的三级结构；以及(5)整合结果以开发和测试催化机制的模型。这项工作的重要性包括促进我们对RNA结构及其如何导致催化活性的理解，催化RNA和RNP在生物反应中的作用，以及通过在基因组研究中确定基因功能和开发基于核酶的选择性遗传和病毒疾病治疗方法，应用我们对催化RNA和核酶工程的理解来改善健康。

An integrated series of biochemical, biophysical, and combinatorial experiments is designed to answer three fundamental questions concerning the hairpin ribozyme: First, how does the RNA assemble into a catalytic structure? Second, what is the structure of the ribozyme-substrate complex? Third, how does this structure result in the acquisition of catalytic activity? The hairpin ribozyme is well suited to such experiments, since the molecule is small enough that comprehensive studies can be undertaken, yet complex enough that significant conformational changes occur during the catalytic cycle. The hairpin differs in several important respects from the only two RNA enzymes that have been more extensively studied, the hammerhead ribozyme and group I introns. Specific Aims are: (1) Map the tertiary structure of the ribozyme-substrate complex; (2) Determine the mechanisms through which metal ions and inhibitors modulate catalytic activity; (3) Define the folding pathway and determine the requirements, kinetics, and thermodynamics of folding and unfolding; (4) Determine the tertiary structure of the ribozyme- substrate complex at high resolution; and (5) Integrate results to develop and test models of the catalytic mechanism. The importance of this work includes advancement of our understanding of RNA structure and how it leads to catalytic activity, the role of catalytic RNA and RNP in biological reactions, and the application of our understanding of catalytic RNA and ribozyme engineering to improving heath, through identifying gene function in genomic investigations and the development of selective ribozyme-based therapeutics for genetic and viral diseases.