HAIRPIN RIBOZYME--FOLDING, STRUCTURE, AND MECHANISM

一系列生物化学、生物物理和组合实验旨在回答关于发夹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-底物复合物的结构是什么？第三，这种结构如何导致催化活性的获得？发夹核酶非常适合这样的实验，因为分子足够小，可以进行全面的研究，但又足够复杂，在催化循环中会发生重大的构象变化。发夹蛋白在几个重要方面不同于仅有的两种被广泛研究的RNA酶，锤头核酶和I组内含子。具体目的有：(1)绘制核酶-底物复合物的三级结构；(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，确定折叠和展开的要求、动力学和热力学；(4)高分辨率测定核酶-底物复合物的三级结构；(5)整合研究结果，建立和测试催化机理模型。这项工作的重要性包括我们对RNA结构及其如何导致催化活性的理解的进步，催化RNA和RNP在生物反应中的作用，以及我们对催化RNA和核酶工程的理解在改善健康方面的应用，通过在基因组研究中识别基因功能和开发基于选择性核酶的遗传性和病毒性疾病治疗方法。

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.