Enzymology of RNA Processing

描述（由申请人提供）：核糖核酸酶P （RNase P）催化前体tRNA （pre-tRNA）的5'端成熟形成tRNA， tRNA是蛋白质合成的重要组成部分。RNase P存在于生命的所有领域，但这种不可或缺的酶的组成各不相同，从细菌中的rna -蛋白质异二聚体到人类线粒体RNase P （mtRNase P）的三种蛋白质复合物。这些酶为定义区分RNA和蛋白质催化的催化特征提供了理想的系统。此外，不同的亚基组成突出了细菌RNase P作为新型抗生素靶点的潜力。在线粒体中，（mt）tRNA和mtRNase P亚基的突变与许多疾病有关，包括神经退行性变、x连锁智力低下、心肌梗死、冠状动脉疾病以及线粒体功能障碍，其临床表现为MELAS（线粒体肌病、脑病、乳酸酸中毒和卒中样症状）、进行性眼外麻痹和/或糖尿病。分析mtRNase P的体内和体外功能将有助于深入了解线粒体tRNA加工途径及其在线粒体生物发生和功能障碍中的作用。因此，对RNase P结构和功能的研究可能对各种健康问题产生广泛的影响，从改善抗菌治疗到表征与多种线粒体疾病发病机制相关的生物学途径。这项建议包括两个主要目标。首先，我们建议发展生物物理方法，包括单分子荧光光谱和核磁共振光谱（与Al-Hashimi教授和Walter教授合作）来研究大RNA分子（如RNase P RNA亚基）的两个标志特征：在扩散、内球和外球接触之间交换的动态RNA-金属相互作用；构象可塑性是RNA功能的核心，包括底物识别和催化。在将这些方法应用于细菌RNase P的过程中，我们的目标是：(1)探索RNase P在整个催化循环中发生的结构和动力学变化；(2)描述RNase P中金属离子结合位点的结构和相互作用。其次，我们将确定新发现的基于蛋白质的mtRNase P所采用的策略来实现催化和底物识别。特别是，我们利用诱变、金属取代和动力学分析来探索MRPP3的功能，以阐明这一预测具有金属依赖性核酸酶活性的新家族成员的机制特征。最后，我们将研究前tRNA识别的决定因素以及mtRNase P加工缺陷在人类线粒体tRNA突变的病理生理机制中的作用。这些研究将大大提高我们对这两种不同类型的RNase P酶及其同源物的结构和功能的理解，为研究类似酶提供有用的方法，并为生物催化的本质提供基本的见解。

DESCRIPTION (provided by applicant): Ribonuclease P (RNase P) catalyzes 5' end maturation of precursor tRNA (pre-tRNA) to form tRNA, an essential component of protein synthesis. RNase P is found in all domains of life, but the composition of this indispensable enzyme varies from a RNA-protein heterodimer in bacteria to a complex of three proteins in human mitochondrial RNase P (mtRNase P). These enzymes provide an ideal system for defining catalytic features that distinguish RNA- and protein-based catalysis. Furthermore, the distinct subunit compositions highlight the potential of bacterial RNase P as a novel antibiotic target. In mitochondria, mutations in (mt)tRNA and mtRNase P subunits have been linked to a number of diseases, including neurodegeneration, X-linked mental retardation, myocardial infarction, coronary artery disease as well as mitochondria dysfunction which manifests clinically as MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like symptoms), progressive external opthalmoplegia and/or diabetes. Analysis of the in vivo and in vitro function of mtRNase P will provide insight into mitochondrial tRNA processing pathways and their role in mitochondria biogenesis and dysfunction. Thus, investigation of RNase P structure and function has the potential for wide-ranging impact on a variety of health issues, from improving antibacterial therapeutics to characterization of the biological pathways linked to the pathogenesis of multiple mitochondrial diseases. This proposal consists of two primary objectives. First, we propose to develop biophysical methods, including single molecule fluorescence spectroscopy and NMR spectroscopy (in collaboration with Professors Al-Hashimi and Walter) to investigate two hallmark features of large RNA molecules, such as the RNase P RNA subunit: dynamic RNA-metal interactions that exchange between diffusive, inner-sphere, and outer- sphere contacts; and conformational plasticity that is central to RNA function, including substrate recognition and catalysis. In applying these methods to bacterial RNase P we aim to: (1) explore the changes in structure and dynamics that occur in RNase P throughout the catalytic cycle; and (2) delineate the structure and interactions within proposed metal ion binding sites in RNase P. Second, we will identify the strategies employed by the newly discovered protein-based mtRNase P to achieve catalysis and substrate recognition. In particular, we explore the function of MRPP3 using mutagenesis, metal substitution and kinetic analysis to elucidate mechanistic features of this member of a novel family predicted to have metal-dependent nuclease activity. Finally, we will examine determinants of pre-tRNA recognition and the role of defects in mtRNase P processing in the pathophysiological mechanisms of human mitochondrial tRNA mutations. These studies will significantly enhance our understanding of the structure and function of these two distinct classes of RNase P enzymes and their homologues, develop methods useful for studying similar enzymes, and provide fundamental insights into the nature of biological catalysis. PUBLIC HEALTH RELEVANCE: RNase P is essential for the formation of tRNA, which plays a key role in protein synthesis. Due to this essential role in life, RNase P has potential medical applications as a novel antibiotic target. Furthermore, errors in tRNA processing in mitochondria are linked to multiple mitochondrial dysfunction diseases. The studies in this proposal will provide insight into the development of inhibitors of bacterial RNase P and yield important information about tRNA processing pathways and the function of mitochondrial RNase P that will impact our understanding of mitochondrial-related disorders.