HAIRPIN RIBOZYME--FOLDING, STRUCTURE, AND MECHANISM

发夹核酶——折叠、结构和机制

• 批准号: 6124227
• 负责人: JOHN MacKenzie BURKE
• 金额: $ 41.56万
• 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 2003-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6124227
• 关键词: catalyst; combinatorial chemistry; enzyme mechanism; enzyme structure; enzyme substrate; nucleic acid structure; protein folding; ribozymes; thermodynamics

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.

一系列集成的生化，生物物理和组合实验旨在回答有关发夹核酶的三个基本问题：首先，RNA如何组装成催化结构？ 其次，核酶 - 基底络合物的结构是什么？ 第三，这种结构如何导致催化活性的获取？发夹核酶非常适合这种实验，因为该分子足够小，可以进行全面的研究，但足够复杂，以至于在催化循环中发生了显着的构象变化。 在几个重要方面，发夹与仅经过更广泛研究的两个RNA酶不同，即锤头核酶和I组内含子。 具体目的是：（1）绘制核酶 - 基底络合物的三级结构； （2）确定金属离子和抑制剂调节催化活性的机制； （3）定义折叠途径并确定折叠和展开的需求，动力学和热力学； （4）确定高分辨率下核酶基质复合物的三级结构； （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我们对RNA结构的理解以及它如何导致催化活性，催化RNA和RNP在生物学反应中的作用，以及我们对催化RNA和核酶工程的了解，通过鉴定基因组合中的基因研究以及基于基于核酶的发展疾病，以及基于基因的基因和培养基，以及基于核对症的发展，以及基于基于基因的基因和病毒。