Catalytic Mechanism of the Hairpin Ribozyme

描述(由申请人提供)：理解RNA催化的机制仍然是一个耐人寻味的挑战，自从最近证明核糖体是核酶以来，这一挑战的意义越来越大。几种核酶催化相同的可逆RNA裂解反应，但采用不同的结构和不同的动力学和催化机制。这些机制的比较提供了对RNA酶可用催化策略的多样性的洞察。发夹状核酶催化独特的pH和金属离子依赖性是核酶可以完全通过利用RNA官能团完成催化的第一个迹象，而不需要金属阳离子与磷酸、核糖或水氧直接配位。到目前为止，很难区分实验操作的结构和催化效果，严重限制了发夹催化机理的研究。最近的两个事态发展在这方面改变了这一领域。首先，解决了发夹状核酶与底物类似物的晶体结构，底物类似物提供了组成活性部位的官能团的第一高分辨率视图。其次，已经开发出发夹状核酶变体，允许在不破坏功能结构的情况下对具有催化作用的重要核苷酸进行修饰。这项拟议的研究将通过阐明特定RNA官能团在催化化学和活性中心结构中的作用，为发夹状核酶的催化机理开发一个详细的模型。首席研究人员将通过检查缺少特定活性部位碱基的核酶变体或被酸碱强度不同的核酶类似物取代的活性部位碱基的反应的pH-速率曲线，来测试核酶官能团是否介导了一般的酸碱催化。初步数据表明，阳离子核酸碱可能对过渡状态形成的负电荷提供静电稳定，或稳定氧阴离子亲核试剂或离开基团。Fedor e博士将使用基本的核酶救援策略，并通过共价掺入不同电离倾向的核碱基类似物来检验这一概念。最后，她将研究活性中心的结构和生化特征如何通过活性基团的位置和方向影响催化。

DESCRIPTION (provided by applicant): Understanding mechanisms of RNA catalysis remains an intriguing challenge, one that has grown in significance since the recent demonstration that the ribosome is a ribozyme. Several ribozymes catalyze the same reversible RNA cleavage reaction but adopt different structures and exploit distinct kinetic and catalytic mechanisms. Comparison of these mechanisms provides insight into the diversity of catalytic strategies that are available to RNA enzymes. The unique pH and metal ion dependencies of hairpin ribozyme catalysis was the first sign that ribozymes can accomplish catalysis exclusively through the use of RNA functional groups with no requirement for direct coordination of metal cations to phosphate, ribose or water oxygens. Until now, the difficulty of distinguishing structural from catalytic effects of experimental manipulations seriously limited studies of the hairpin catalytic mechanism. Two recent developments have transformed the field in this regard. First, a crystal structure has been solved for a hairpin ribozyme complex with a substrate analog that provides the first high-resolution view of the functional groups that comprise the active site. Second, hairpin ribozyme variants have been developed that allow catalytically important nucleotides to be modified without disrupting the functional structure. The proposed research will develop a detailed model for the hairpin ribozyme catalytic mechanism by elucidating the roles of specific RNA functional groups in catalytic chemistry and active site architecture. The principal investigator will test whether ribozyme functional groups mediate general acid base catalysis by examining pH-rate profiles for reactions of ribozyme variants missing specific active site nucleobases or with active site nucleobases replaced by nucleobase analogs that differ in acid or base strength. Preliminary data suggest that cationic nucleobases might provide electrostatic stabilization to negative charge developing in the transition state or stabilize oxyanion nucleophiles or leaving groups. Dr. Fedor e will examine this notion using an abasic ribozyme rescue strategy and through covalent incorporation of nucleobase analogs that differ in the propensity to undergo ionization. Finally, she will examine how structural and biochemical features of the active site influence catalysis through positioning and orientation of reactive groups.