Enzymology of RNA Processing Enzymes

RNA加工酶的酶学

• 批准号: 7161780
• 负责人: CAROL A FIERKE
• 金额: $ 25.56万
• 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 2009-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7161780
• 关键词: Active Sites; Affinity; Amino Acids; Antibiotics; Bacillus anthracis; Bacteria; Base Pairing; Binding; Binding Sites; Biochemical; Catalysis; Catalytic Domain; Catalytic RNA; Collaborations; Complex; Coupling; Data; Development; Docking; Endoribonucleases; Enzymatic Biochemistry; Enzymes; Eukaryota; Eukaryotic Cell; Evolution; Fluorescence; Fluorescence Resonance Energy Transfer; Glycine decarboxylase; Goals; Grant; Helix (Snails); Holoenzymes; Hydrogen Bonding; In Vitro; Ions; Kinetics; Measurement; Measures; Medical; Metal Binding Site; Metal Ion Binding; Metals; Methods; Molecular Conformation; Mutagenesis; NMR Spectroscopy; Organism; Pancreatic ribonuclease; Positioning Attribute; Property; Protein Biosynthesis; Protein Subunits; Proteins; RNA; RNA Processing; RNA-Binding Proteins; RNase P; Range; Relative (related person); Research Personnel; Research Project Grants; Residual state; Ribonucleases; Role; Site; Staphylococcus aureus; Structural Models; Structure; Substrate Interaction; Techniques; Thermodynamics; Time; Transfer RNA; catalyst; crosslink; ear helix; in vivo; inhibitor/antagonist; insight; molecular recognition; mutant; novel; nuclear Overhauser enhancement; programs; restraint; structural biology; success; tRNA Precursor; time use

DESCRIPTION (provided by applicant): Ribonuclease P (RNase P) catalyzes the maturation of the 5' end of precursor tRNA (pre-tRNA) to form tRNA, a component essential for the synthesis of proteins. RNase P from bacteria is composed of an RNA subunit that catalyzes pre-tRNA cleavage in vitro and a protein component that is essential for activity in vivo and enhances binding of the pre-tRNA substrate. In contrast, RNase P in eukaryotes contains one RNA and multiple protein subunits. We propose to investigate the function of the bacterial RNase P using a combination of biochemical and structural techniques. Specifically, we aim to: (1) explore the structure and dynamics of RNase P using time resolved fluorescence resonance energy transfer techniques to measure distances and mobility; (2) investigate substrate recognition in bacterial RNas P by determining the thermodynamics and function of PRNA-pre-tRNA and pre-tRNA-P protein contacts in RNase P and investigating the cleavage of novel RNA substrates; (3) investigate the position and functions of metals bound to RNase P for both catalysis and substrate recognition; and (4) delineate the position of metal ion binding sites in RNase P and the structure of isolated helices by NMR spectroscopic analysis. Our long term goal is to further understand (1) the mechanisms of catalysis used by ribozymes as compared to protein enzymes, and (2) the structure and energetics of RNA binding proteins and protein/RNA complexes. RNase P is a unique enzyme to investigate catalytic strategies and substrate recognition since the active site is near the protein-RNA interface. This unique collaboration between the protein and RNA subunits may provide insight into the evolution from RNA to protein catalysts. RNase P is an essential enzyme as tRNA maturation is required for protein synthesis. RNase P has potential medical applications as a novel antibiotic target since it is an essential enzyme and the eukaryotic and prokaryotic enzymes have different subunit composition. The structural and functional studies proposed here should provide insight into the development of active site-directed inhibitors of bacterial RNase P from target organisms such as S. aureus and Bacillus anthracis.

描述（申请人提供）： 核糖核酸酶 P (RNase P) 催化前体 tRNA (pre-tRNA) 5' 端成熟，形成 tRNA，tRNA 是合成蛋白质所必需的成分。来自细菌的 RNase P 由在体外催化前 tRNA 裂解的 RNA 亚基和对体内活性至关重要并增强前 tRNA 底物结合的蛋白质成分组成。相比之下，真核生物中的 RNase P 包含一种 RNA 和多种蛋白质亚基。我们建议结合生化和结构技术来研究细菌 RNase P 的功能。具体来说，我们的目标是：（1）利用时间分辨荧光共振能量转移技术来测量距离和迁移率，探索 RNase P 的结构和动力学； (2) 通过测定 RNase P 中 PRNA-pre-tRNA 和 pre-tRNA-P 蛋白接触的热力学和功能，并研究新型 RNA 底物的裂解，研究细菌 RNas P 中的底物识别； (3) 研究与 RNase P 结合的金属在催化和底物识别中的位置和功能； (4)通过核磁共振波谱分析描绘RNase P中金属离子结合位点的位置和分离螺旋的结构。我们的长期目标是进一步了解 (1) 核酶与蛋白酶相比的催化机制，以及 (2) RNA 结合蛋白和蛋白质/RNA 复合物的结构和能量学。 RNase P 是一种独特的酶，用于研究催化策略和底物识别，因为其活性位点靠近蛋白质-RNA 界面。蛋白质和 RNA 亚基之间的这种独特合作可能有助于深入了解从 RNA 到蛋白质催化剂的进化。 RNase P 是一种必需酶，因为蛋白质合成需要 tRNA 成熟。 RNase P 作为一种新型抗生素靶点具有潜在的医学应用，因为它是一种必需酶，并且真核酶和原核酶具有不同的亚基组成。这里提出的结构和功能研究应该有助于深入了解来自金黄色葡萄球菌和炭疽芽孢杆菌等目标生物体的细菌 RNase P 活性定点抑制剂的开发。