Structure and function of RNase P

描述(由申请人提供)：我们对RNA结构的了解有了爆炸性的增长，在确定RNA在基因表达和调控中的作用方面取得了突破性的进展。然而，我们对决定其生物学功能的RNA的化学和生物物理特性的了解进展不那么快。这一缺陷是由于问题的复杂性，但也是因为缺乏足够的实验工具来揭示机械细节。核糖核蛋白酶核糖核酸酶P(RNase P)催化tRNA前体(PtRNAs)的5‘端成熟，已成为了解包括催化在内的RNA结构和功能的一个简单而广泛的系统。我们项目的长期目标是在化学细节水平上了解RNaseP核糖核蛋白是如何实现其巨大的速率增强和多底物特异性的。对这两个过程都至关重要的是镁离子的位点特异性结合。在我们对RNaseP的实验分析中集成了创新的研究工具，旨在克服三个领域的关键实验限制：定义RNA-金属离子相互作用(拉曼光谱)；识别催化相互作用(动力学同位素效应)；以及理解多底物识别(高通量测序)。像许多酶一样，核糖核酸酶P在细胞中处理多种不同的底物。这一性质提出了一个普遍的问题，即酶如何区分同源和非同源底物，以及它如何适应不同底物之间结构的变化。我们正在比较不同ptRNA加工反应的动力学，并应用一种新的高通量方法来确定控制最佳催化效率的ptRNA序列P。尽管进行了大量的研究，但核酶所采用的催化模式，包括RNaseP，在实验上还没有被很好地理解或表征。我们正在进行详细的机制分析，通过观察P RNA和ptRNA中特定位点的官能团修饰如何影响过渡态的电荷分布，来测试所提出的活性位点相互作用。溶液中的镁离子之间的相互作用对于所有RNA的功能都是必不可少的，建立单个离子或离子类别之间的结合关系是一个非常感兴趣的领域。然而，像大多数RNA一样，P RNA中单个离子相互作用与结合和催化的关键酶功能之间的联系还不是很清楚。在最后一个项目期间，我们开发了一种使用拉曼光谱来检测和量化金属离子与RNA磷酸的相互作用的方法。为了跟进这些进展，我们正在使用拉曼光谱和直接离子缔合测量来检测ES络合物形成时离子的吸收，并测试P4残基在离子结合中的作用。此外，我们正在开发使用同位素编辑的拉曼检测单个磷酸盐上的离子结合的方法。

DESCRIPTION (provided by applicant): There has been an explosive increase in our understanding of RNA structure and ground breaking advances in defining the roles of RNAs in gene expression and regulation. Yet, our understanding of the chemical and biophysical properties of RNA that determine its biological function has advanced less quickly. This deficiency is due to the complexity of the problem, but also due to the lack of sufficient experimental tools for revealing mechanistic detail. The ribonucleoprotein enzyme ribonuclease P (RNase P), which catalyzes the essential 5' end maturation of tRNA precursors (ptRNAs), has emerged as an elegantly simple and broadly useful system to understand RNA structure and function including catalysis. The long term goal of our project is to understand, at a chemical level of detail, how the RNase P ribonucleoprotein achieves its enormous rate enhancement and its multiple substrate specificity. Essential to both processes is site-specific binding of Mg2+ ions. Integrated into our experimental analyses of RNase P are innovative research tools designed to overcome key experimental limitations in three areas: defining RNA-metal ion interactions (Raman spectroscopy); identifying catalytic interactions (kinetic isotope effects); and understanding multiple substrate recognition (high-throughput sequencing). RNase P, like many enzymes, processes multiple different substrates in the cell. This property raises the general problem of how the enzyme distinguishes between cognate and non-cognate substrates and how it accommodates the variation in structure between different substrates. We are comparing the kinetics of different ptRNA processing reactions, and applying a novel high-throughput method to identify the ptRNA sequences P that control optimal catalytic efficiency. Despite intense investigation, the catalytic modes employed by ribozymes, including RNase P, are not well understood or characterized experimentally. We are pursuing detailed mechanistic analyses to test proposed active site interactions by observing how site-specific functional group modifications in P RNA and ptRNA influence the charge distribution in the transition state. The interaction of solution Mg2+ ions is essential for the function of all RNAs, and establishing the relationships between the binding of individual ions or classes of ions is an area of intense interest. However, like most RNAs the linkages between individual ion interactions in P RNA, and the critical enzyme functions of binding and catalysis are not well understood. In the last project period, we developed a means to detect and quantify metal ion interactions with RNA phosphates using Raman spectroscopy. To follow up on these advances we are using Raman spectroscopy and direct ion association measurements to detect the uptake of ions upon formation of the ES complex and to test the roles of P4 residues in ion binding. Additionally we are developing methods to detect ion binding at individual phosphates using isotope-edited Raman.