Hammerhead Ribozyme: Active Site Assembly and Structure

描述(由申请人提供)：尽管对锤头进行了深入的分析 核酶生物化学及其活性两种晶体结构的阐明 位置结构和反应机理尚不清楚。相比之下，在 发夹状核酶系统提供了更清晰的活性部位 组装，并导致了开发和实验验证 结构模型和机械模型。建议的工作是基于两个可测试的 假设。首先，伯克博士提出，现有的晶体结构 锤头状核酶代表一种类似于非对接结构的状态 发夹状核酶，并有类似的显著构象变化 发夹状核酶对接是锤头组装中必不可少的一步 活性部位，是卵裂的必备前奏。第二，最近 伯克博士实验室中关于锤头系统的实验工作使他 提出锤头状核酶和发夹状核酶具有共同的特征 活性中心结构和反应机理，锤头G12 核酶可能在结构和功能上与G8相似，G8是一种关键的催化物质 发夹状核酶催化碱基。该提案的具体目标是：(1) 确定组装活性部位所需的构象变化，(2) 确定锤头活性部位的基本成分，以及(3)开发 并对活性中心的结构和机理模型进行了测试。在这些研究中， 伯克博士和他的团队拥有的大量实验方法 为他们研究发夹状核酶将是非常有价值的。 这一结果将为我们提供有关分子进化的重要见解。 生物催化剂和核糖核酸和核糖核酸的机理策略 核糖核蛋白酶，并将直接促进核酶的使用 在功能基因组学和基因治疗方面。

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.