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有趣的挑战，自从最近证明核糖体是核酶以来，这一挑战变得越来越重要。几种核酶催化相同的可逆RNA切割反应，但采用不同的结构，并利用不同的动力学和催化机制。这些机制的比较提供了深入了解的多样性，可用于RNA酶的催化策略。发夹核酶催化的独特pH和金属离子依赖性是核酶可以仅通过使用RNA官能团而不需要金属阳离子与磷酸根、核糖或水氧直接配位来实现催化的第一个迹象。到目前为止，区分实验操作的结构和催化效应的困难严重限制了发夹催化机制的研究。在这方面，最近的两个事态发展改变了这一领域。首先，已经解决了发夹核酶复合物的晶体结构与底物类似物，提供了第一个高分辨率的功能团，包括活性位点的视图。第二，已经开发了发夹核酶变体，其允许在不破坏功能结构的情况下修饰催化重要的核苷酸。该研究将通过阐明特定RNA功能基团在催化化学和活性位点结构中的作用，为发夹状核酶的催化机制建立一个详细的模型。主要研究者将通过检查缺失特定活性位点核碱基或活性位点核碱基被酸或碱强度不同的核碱基类似物取代的核酶变体的反应的pH速率曲线来测试核酶官能团是否介导一般的酸碱催化。初步数据表明，阳离子核碱基可能提供静电稳定负电荷发展中的过渡态或稳定含氧阴离子亲核试剂或离去基团。Fedor e博士将使用无碱基核酶拯救策略并通过共价掺入不同电离倾向的核碱基类似物来研究这一概念。最后，她将研究如何通过定位和反应基团的方向的活性部位的结构和生化特征影响催化。

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.