Hammerhead Ribozyme: Active Site Assembly and Structure

描述（由申请人提供）：尽管对锤头鲨进行了深入分析， 核酶的生物化学和两种晶体结构的阐明， 位点结构和反应机理仍然未知。与此相反， 发夹状核酶系统提供了活性位点更清晰视图 组装，并导致了开发和实验验证， 结构和机械模型。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 发夹状核酶的一个显著的构象变化类似于 与发夹状核酶的对接是锤头状核酶组装的重要步骤 活性位点，并且是裂解的强制性前奏。第二，近期 伯克博士在实验室里对锤头系统的实验工作使他 提出锤头状和发夹状核酶具有共同的特征， 活性中心结构和反应机理，以及锤头G12 核酶可能在结构和功能上类似于G8，G8是一种关键的催化酶， 发夹状核酶催化中的碱基。建议的具体目标是：（1） 确定组装活性位点所需的构象变化，（2） 确定锤头活性部位的基本组成部分，以及（3）开发 以及活性部位结构和机制的测试模型。在这些研究中， Burke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而开发的技术将是极其有价值的。 这些结果将提供关键的见解分子进化的 生物催化剂和RNA所采用的机械策略， 核糖核蛋白酶，并将直接有助于使用核酶 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.