Enzymology of RNA Processing

RNA 加工的酶学

基本信息

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
项目类别：
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

项目摘要

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（由申请人提供）：核糖核酸酶P（RNase P）催化5'前体tRNA（pre-tRNA）的末端成熟以形成tRNA，这是蛋白质合成的重要组成部分。 RNase P在生命的所有领域中都发现，但是这种必不可少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）中三种蛋白的复合物不同。这些酶为定义区分基于RNA和蛋白质的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作为新型抗生素靶标的潜力。 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类似于中风的症状），进行性外科治疗术和/或糖尿病。分析MTRNase P的体内和体外功能将提供有关线粒体TRNA处理途径及其在线粒体生物发生和功能障碍中的作用的见解。因此，对RNase P结构和功能的研究可能会对各种健康问题产生广泛影响，从改善抗菌疗法到与多种线粒体疾病的发病机理相关的生物学途径的表征。该提案由两个主要目标组成。首先，我们建议开发生物物理方法，包括单分子荧光光谱和NMR光谱法（与教授Al-Hashimi和Walter合作），以研究大型RNA分子的两个标志性特征，例如RNase P RNA亚基：动力学RNA亚基：动态RNA - 米特相互作用，这些相互作用在差异，sphere-spheere之间，以及外在的 - 外在 - 外在 - 外在 - 外在 - 外在 - 外在 - 外在 - 外部 - 外部 - 外部 - 外部 - 外部 - 外部 - 外部 - 外部 - 外部 - 外部 - 外部 - 外部 - 外部 - 外部 - 外部 - 外部 - 互动;和RNA功能核心的构象可塑性，包括底物识别和催化。在将这些方法应用于细菌RNase P时，我们的目的是：（1）探索整个催化循环中RNase P中发生的结构和动力学的变化；（2）描述RNase P中提出的金属离子结合位点内的结构和相互作用。第二，我们将确定新发现的基于蛋白质的mtrnase P所采用的策略，以实现催化和底物识别。特别是，我们使用诱变，金属取代和动力学分析探讨了MRPP3的功能，以阐明该新家族中该成员的机械特征，预测具有金属依赖性的核酸酶活性。最后，我们将检查pre-tRNA识别的决定因素以及缺陷在MTRNASE P加工中的作用在人线粒体TRNA突变的病理生理机理中。这些研究将显着增强我们对这两种不同类别的RNase P酶及其同源物的结构和功能的理解，开发用于研究相似酶的方法，并提供对生物催化性质的基本见解。