Structure/Function of Ribonuclease P

描述（由申请人提供）：已充分确定酶进行对人体细胞功能至关重要的化学反应。我们现在知道，几种关键酶，包括进行蛋白质合成的核糖体，都含有直接参与催化作用的必需RNA分子。我们研究计划的长期目标是了解由RNA组成的酶活性位点如何实现与蛋白酶相当的特异性和化学速率增强。为了实现这一目标，我们采用了催化RNA，或核酶，来自细菌核糖核酸酶P。这种核酶是高度保守的，并在人类中发现的酶同源。因此，它为探索RNA结构和功能的基本方面提供了一个很好的实验系统。RNase P使用金属离子来促进水分子的攻击，以破坏RNA前体中的特定磷酸二酯键，从而产生功能性tRNA分子。拟议的研究旨在提供对酶机制的理解，金属离子在催化中的特定作用，以及非共价相互作用如何有助于特异性和速率增强的功能描述。通过研究重同位素取代参与化学反应的原子的效果，将深入了解催化机理。我们将开始与攻击水的分析，并在长期内，扩大这些研究，包括其他原子参与反应。我们将通过研究取代RNA活性位点内的特定原子对反应速率和金属离子依赖性的影响来确定催化金属离子结合的位点。非共价结合相互作用的作用将通过测试特定的模型，通过对核酶及其底物进行补偿突变的相互作用进行探索。此外，由于酶的构象通常是动态的，我们将扩展这些研究，以确定构象的变化如何与特异性和催化作用联系起来。所解决的机制问题对所有酶都是共同的，因此我们期望所获得的信息将增加我们对生物催化的理解。此外，核酶，包括RNase P，已经适应于治疗目的，我们将获得的结果应该有助于将催化RNA应用于这一目标。

DESCRIPTION (provided by applicant): It is well established that enzymes carry out the chemical reactions that are essential to human cell function. We now know that several key enzymes, including the ribosome that carries out protein synthesis, contain essential RNA molecules that participate directly in catalysis. The long-term goal of our research program is to understand how an enzyme active site composed of RNA achieves specificity and chemical rate enhancement comparable to protein enzymes. To pursue this objective we have employed the catalytic RNA, or ribozyme, derived from bacterial ribonuclease P. This ribozyme is highly conserved and homologous to the enzyme found in humans. Thus, it provides an excellent experimental system for exploring fundamental aspects of RNA structure and function. RNase P uses metal ions to promote the attack of a water molecule to break a specific phosphodiester linkage in an RNA precursor to generate a functional tRNA molecule. The proposed research is aimed at providing an understanding of enzyme mechanism, the specific role of metal ions in catalysis, and a functional description of how non-covalent interactions contribute to specificity and rate enhancement. Insight into catalytic mechanism will be gained by examining the effect of substituting heavy isotopes for atoms involved in the chemical reaction. We will begin with an analysis or the attacking water and, in the long term, extend these studies to encompass additional atoms involved in the reaction. We will identify sites of catalytic metal ion binding by examining the effects of substituting specific atoms within the active site of the RNA on reaction rate and metal ion dependence. The role of non-covalent binding interactions will be explored by testing specific models for interactions by making compensatory mutations on the ribozyme and its substrate. Additionally, because enzymes conformation of is generally dynamic, we will extend these studies to determine how changes in conformation are linked to specificity and catalysis. The issues of mechanism addressed are common to all enzymes, thus we expect that the information gained will add to our understanding of biological catalysis. Furthermore, ribozymes, including RNase P, have been adapted to therapeutic purposes, and the results we will obtain should assist in applying catalytic RNAs to this goal.