Hammerhead Ribozyme: Active Site Assembly and Structure

锤头核酶：活性位点组装和结构

• 批准号: 6712112
• 负责人: JOHN MacKenzie BURKE
• 金额: $ 28.03万
• 依托单位: UNIVERSITY OF VERMONT & ST AGRIC COLLEGE
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-04-01 至 2007-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6712112
• 关键词: active sites; biochemical evolution; chemical models; chemical structure function; conformation; model design /development; molecular assembly /self assembly; nucleic acid structure; ribozymes

DESCRIPTION (provided by applicant): Despite intensive analysis of hammerhead ribozyme biochemistry and the elucidation of two crystal structures, the active site structure and reaction mechanism remain unknown. In contrast, work in the hairpin ribozyme system has provided a much clearer view of active site assembly, and has led to the development and experimental verification of structural and mechanistic models. The proposed work is based on two testable hypotheses. First, Dr. Burke proposes that the existing crystal structures of the hammerhead ribozyme represent a state analogous to the undocked structure of the hairpin ribozyme, and that a significant conformational change analogous to hairpin ribozyme docking is an essential step in assembly of the hammerhead active site, and is an obligatory prelude to cleavage. Second, recent experimental work on the hammerhead system in Dr. Burke's laboratory leads him to propose that the hammerhead and hairpin ribozymes share common features of active site structure and reaction mechanism, and that G12 of the hammerhead ribozyme may be structurally and functionally analogous to G8, a key catalytic base in hairpin ribozyme catalysis. Specific Aims of the proposal are: (1) Identify conformational changes required to assemble the active site, (2) Identify essential components of the hammerhead active site, and (3) Develop and test models for active site structure and mechanism. In these studies, the extensive repertoire of experimental methods that Dr. Burke and his group have developed for their studies of hairpin ribozymes will be extremely valuable. The results will provide critical insights into the molecular evolution of biological catalysts and the mechanistic strategies employed by RNA and ribonucleoprotein enzymes, and will directly contribute to the use of ribozymes in functional genomics and gene therapy.

描述（由申请人提供）：尽管对Hammerhead进行了深入分析 核酶生物化学和两个晶体结构阐明活性 位点结构和反应机制仍然未知。相反，在 发夹核酶系统提供了更清晰的活性场所的视图 组装，并导致了开发和实验验证 结构和机械模型。拟议的工作基于两个可测试 假设。首先，伯克博士提出现有的晶体结构 锤头核酶代表类似于不可锁定结构的状态 发夹核酶，以及显着的构象变化类似 发夹核酶对接是锤头组装的重要步骤 主动部位，是裂解的强制性前奏。第二，最近 Burke博士实验室中的Hammerhead系统的实验性工作导致他 提出锤头和发夹核酶具有共同的特征 主动位点结构和反应机制，以及锤头的G12 核酶可能在结构和功能上类似于g8，一种钥匙催化 基于发夹核酶催化的基础。该提案的具体目的是：（1） 确定组装活性位点所需的构象变化，（2） 确定锤头主动站点的基本组件，（3）发展 和主动部位结构和机制的测试模型。在这些研究中， Burke博士和他的小组拥有的广泛的实验方法曲目 开发用于对发夹核酶的研究将非常有价值。 结果将提供有关分子演化的关键见解 RNA和 核糖核蛋白酶，并将直接有助于核酶的使用 在功能基因组学和基因治疗中。