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（由申请人提供）：核糖核酸酶 P (RNase P) 催化前体 tRNA (pre-tRNA) 的 5' 末端成熟，形成 tRNA，tRNA 是蛋白质合成的重要组成部分。 RNase P 存在于生命的各个领域，但这种不可或缺的酶的组成各不相同，从细菌中的 RNA 蛋白异二聚体到人类线粒体 RNase P (mtRNase P) 中的三种蛋白的复合物。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，不同的亚基组成凸显了细菌 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，包括神经变性、X 连锁智力低下、心肌梗死、冠状动脉疾病以及临床表现为 MELAS 的线粒体功能障碍（线粒体肌病、脑病、乳酸性酸中毒和 中风样症状）、进行性外眼肌麻痹和/或糖尿病。对 mtRNase P 的体内和体外功能的分析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 结构和功能的研究有可能对各种健康问题产生广泛影响，从改进抗菌治疗到表征与多种线粒体疾病发病机制相关的生物途径。 该提案有两个主要目标。首先，我们建议开发生物物理方法，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 分子的两个标志特征，例如 RNase P RNA 亚基：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。将这些方法应用于细菌 RNase P 时，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 中提议的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探索了 MRPP3 的功能，以阐明该新家族成员的机制特征，预计具有金属依赖性核酸酶活性。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。这些研究将显着增强我们对这两类不同的 RNase P 酶及其同系物的结构和功能的理解，开发可用于研究类似酶的方法，并为生物催化的本质提供基本见解。