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（由申请人提供）：核糖核酸酶P（RNase P）催化前体TRNA（pre-tRNA）的5'末端形成tRNA，这是蛋白质合成必不可少的成分。来自细菌的RNase P由RNA亚基组成，该亚基在体外催化前基因裂解和蛋白质成分对于体内活性至关重要，并增强了pre-tRNA底物的结合。相反，真核生物中的RNase P包含一个RNA和多个蛋白质亚基。我们建议使用生化和结构技术的组合研究细菌RNase P的功能。具体而言，我们的目的是：（1）使用时间分辨的荧光共振能量传递技术探索RNase P的结构和动力学，以测量距离和迁移率； （2）通过确定RNase P中PRNA-PRE-TRNA和PRNA-P蛋白接触的热力学和功能，研究细菌RNA P中的底物识别，并研究新型RNA底物的裂解； （3）研究与RNase P结合的金属的位置和功能，用于催化和底物识别； （4）通过NMR光谱分析描绘了RNase P中金属离子结合位点的位置以及分离的螺旋结构。我们的长期目标是进一步理解（1）与蛋白质酶相比，核酶使用的催化机制，以及（2）RNA结合蛋白和蛋白/RNA复合物的结构和能量。 RNase P是研究催化策略和底物识别的独特酶，因为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的合作可能会洞悉从RNA到蛋白质催化剂的演变。 RNase P是必不可少的酶，因为蛋白质合成需要tRNA成熟。 RNase P具有潜在的医学应用作为一种新型的抗生素靶标，因为它是一种必不可少的酶，而真核和原核酶具有不同的亚基组成。此处提出的结构和功能研究应洞悉从靶向生物（如金黄色葡萄球菌和炭疽芽孢杆菌）的细菌RNase P的活性定位抑制剂的发展。