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一种核酶以来，这一挑战变得越来越重要。几种核酶催化相同的可逆RNA裂解反应，但采用不同的结构，具有不同的动力学和催化机理。这些机制的比较提供了对RNA酶可用的催化策略的多样性的见解。发夹核酶催化的独特pH值和金属离子依赖性是核酶可以完全通过使用RNA官能团来完成催化而不需要金属阳离子与磷酸、核糖或水氧直接配位的第一个标志。到目前为止，难以区分实验操作的结构和催化效应严重限制了对发夹催化机理的研究。在这方面，最近的两项发展改变了这一领域。首先，一种带有底物类似物的发夹核酶复合体的晶体结构已经得到解决，该结构提供了构成活性位点的官能团的第一个高分辨率视图。其次，发夹核酶变体已经被开发出来，它允许在不破坏功能结构的情况下修饰具有催化作用的重要核苷酸。本研究将通过阐明特定RNA官能团在催化化学和活性位点结构中的作用，建立发夹核酶催化机制的详细模型。主要研究者将通过检查缺少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异体反应的ph速率谱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，阳离子核碱基可能对过渡态形成的负电荷起到静电稳定作用，或稳定氧阴离子亲核试剂或离去基。费多尔博士将使用一种基本的核酶拯救策略，并通过共价结合核碱基类似物来检验这一概念，这些核碱基类似物在电离倾向上有所不同。最后，她将研究活性位点的结构和生化特征如何通过反应基团的定位和取向影响催化。

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.