Enzymology of RNA Processing

RNA 加工的酶学

• 批准号: 8402158
• 负责人: CAROL A FIERKE
• 金额: $ 29.03万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-01-01 至 2014-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8402158
• 关键词: Active Sites; Anti-Bacterial Agents; Antibiotics; Bacteria; Binding; Binding Sites; Biogenesis; Biological; Biological Models; Biological Process; Cardiovascular system; Catalysis; Catalytic RNA; Chemicals; Collaborations; Complex; Coronary Arteriosclerosis; Coupled; Defect; Development; Diabetes Mellitus; Disease; Docking; Encephalopathies; Energy Transfer; Environment; Enzymatic Biochemistry; Enzymes; Evolution; Family; Fluorescence; Fluorescence Spectroscopy; Functional disorder; Genetic; Glycine decarboxylase; Goals; Grant; Health; Homologous Gene; Human; Hydroxyl Radical; In Vitro; Investigation; Ions; Kinetics; Lactic Acidosis; Lead; Life; Ligand Binding; Ligands; Light; Link; Medical; Metal Binding Site; Metal Ion Binding; Metals; Methods; Mitochondria; Mitochondrial Diseases; Mitochondrial Myopathies; Molecular; Molecular Conformation; Mutagenesis; Mutation; Myocardial Infarction; NMR Spectroscopy; Nature; Nerve Degeneration; Pathogenesis; Pathway interactions; Play; Process; Protein Biosynthesis; Protein Precursors; Protein Subunits; Proteins; RNA; RNA Processing; RNase P; Reaction; Reading; Research; Ribonucleoproteins; Role; Site; Solutions; Stroke; Structure; Substrate Interaction; Substrate Specificity; Symptoms; System; Techniques; Tertiary Protein Structure; Therapeutic; Time; Transfer RNA; X-Linked Mental Retardation; base; cofactor; improved; in vivo; inhibitor/antagonist; innovation; insight; member; mitochondrial dysfunction; molecular recognition; novel; nuclease; phosphodiester; professor; public health relevance; single molecule; success; tRNA Precursor; therapeutic target

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.

描述(申请人提供)：核糖核酸酶P(RNaseP)催化前体tRNA(前-tRNA)的5‘端成熟形成tRNA，这是蛋白质合成的重要组成部分。RNase P存在于生命的各个领域，但这种不可或缺的酶的组成从细菌中的RNA-蛋白质异二聚体到人线粒体RNase P(MtRNase P)中的三种蛋白质的复合体。这些酶提供了一个理想的系统来定义区分基于RNA和基于蛋白质的催化的催化特征。此外，不同的亚基组成突出了细菌RNaseP作为新的抗生素靶点的潜力。在线粒体中，(Mt)tRNA和mtRNase P亚单位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，临床表现为MELAS(线粒体肌病、脑病、乳酸酸中毒和中风样症状)、进行性外眼肌瘫痪和/或糖尿病。对mtRNase P体内和体外功能的分析将有助于深入了解线粒体tRNA的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，对核糖核酸酶P结构和功能的研究有可能对各种健康问题产生广泛的影响，从改进抗菌治疗到表征与多种线粒体疾病的发病机制有关的生物途径。这项提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和Walter教授合作)来研究大RNA分子的两个标志性特征，例如RNaseP RNA亚单位：动态RNA-金属相互作用，在扩散、内球和外球接触之间交换；以及构象可塑性，对RNA功能至关重要，包括底物识别和催化。在将这些方法应用于细菌RNaseP时，我们的目标是：(1)探索RNaseP在整个催化循环中发生的结构和动力学变化；(2)描述RNaseP中建议的金属离子结合部位的结构和相互作用。其次，我们将确定新发现的基于蛋白质的mtRNaseP实现催化和底物识别所采用的策略。特别是，我们通过突变、金属取代和动力学分析来探索MRPP3的功能，以阐明这个被预测具有金属依赖核酸酶活性的新家族的机制特征。最后，我们将检查前tRNA识别的决定因素和mtRNase P处理中缺陷在人类线粒体tRNA突变的病理生理机制中的作用。这些研究将极大地提高我们对这两类不同的RNaseP酶及其同系物的结构和功能的理解，发展有助于研究类似酶的方法，并为了解生物催化的性质提供基本的见解。