Structure and Function in Catalytic RNP Assembly

催化 RNP 组装的结构和功能

• 批准号: 7393785
• 负责人: MARK P. FOSTER
• 金额: $ 27.29万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-04-01 至 2011-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7393785
• 关键词: Archaea; Bacterial Proteins; Binding; Binding Sites; Biochemical; Catalysis; Catalytic RNA; Chemicals; Cleaved cell; Complex; Condition; Data; Derivation procedure; Engineering; Enzymes; Exhibits; Fostering; Glycine decarboxylase; Holoenzymes; Homologous Gene; Human; In Vitro; Individual; Investigation; Life; Light; Maps; Mass Spectrum Analysis; Methanothermobacter; Modeling; Molecular; Molecular Conformation; Organism; Pathway interactions; Phylogenetic Analysis; Protein Binding; Protein Subunits; Proteins; RNA; RNA Conformation; RNA Folding; RNA-Protein Interaction; RNase P; Research Personnel; Ribonucleoproteins; Role; Site; Solutions; Structure; Transfer RNA; Work; base; cofactor; data integration; enzyme substrate complex; gene therapy; in vivo; insight; programs; protein folding; reconstitution; research study; tRNA Precursor; three-dimensional modeling; tool

DESCRIPTION (provided by applicant): The broad, long-term objective of this proposal is to gain structural and dynamic insights into how proteins modulate RNA structure and function in a catalytic ribonucleoprotein (RNP) complex. Ribonuclease P (RNase P) is an essential and ubiquitous ribonucleoprotein enzyme primarily responsible for cleaving the 5' leader sequence during maturation of tRNAs. The eubacterial enzyme is an RNP complex made up of one RNA and one protein subunit. The RNA subunit is catalytic on its own, but the protein is required in vivo. In contrast, several protein subunits with unknown function constitute the human enzyme, and its RNA component alone appears to be inactive. The investigators propose that a principal role of the protein subunits is to stabilize the active conformation of the RNA, and that holoenzyme assembly and substrate recognition proceed via hierarchical mutual binding-induced folding of the protein and RNA subunits. Using the RNase P enzyme from a thermophilic archaebacterium as a model, the investigators will use biochemical and biophysical tools to gain structural insights into the interaction between individual protein components, free and in complex, with the catalytic RNA subunit. The aims are to: 1. Determine the solution structures of one or more protein subunits (or their fragments) by NMR. 2. Map the secondary structure and protein binding sites of the RNA subunit using enzymatic and chemical probes. 3. Characterize protein-protein and protein-RNA interactions in the native and in vitro reconstituted enzyme, and examine whether conformational changes (induced fit) accompany RNP assembly. Successful completion of these aims will enable integration of the data into a three-dimensional model of the RNase P holoenzyme and provide valuable insights into how proteins modulate the enzyme's fUnction. With recent efforts to make use of the substrate selectivity of RNase P to engineer customized ribozymes for use in gene therapy, insights into the molecular basis for the enzyme's function is clearly essential.

描述（由申请人提供）：该提案的广泛长期目标是获得蛋白质如何调节RNA结构和催化核糖核蛋白（RNP）复合物功能的结构和动态见解。核糖核酸酶P（Ribonuclease P，RNase P）是一种广泛存在的核糖核蛋白酶，主要负责在tRNA成熟过程中切割5'端前导序列。真细菌酶是一种RNP复合物，由一个RNA和一个蛋白质亚基组成。RNA亚基本身是催化剂，但蛋白质是体内所需的。相反，几个功能未知的蛋白质亚基构成了人类酶，其RNA组分单独似乎是无活性的。研究人员提出，蛋白质亚基的主要作用是稳定RNA的活性构象，全酶组装和底物识别通过蛋白质和RNA亚基的分级相互结合诱导的折叠进行。使用来自嗜热古细菌的RNase P酶作为模型，研究人员将使用生物化学和生物物理工具来获得对单个蛋白质组分（游离和复合物）与催化RNA亚基之间相互作用的结构见解。目的是：1.通过NMR确定一个或多个蛋白质亚基（或其片段）的溶液结构。2.使用酶和化学探针绘制RNA亚基的二级结构和蛋白质结合位点。3.表征天然和体外重组酶中的蛋白质-蛋白质和蛋白质-RNA相互作用，并检查构象变化（诱导拟合）是否伴随RNP组装。这些目标的成功完成将使数据集成到RNase P全酶的三维模型中，并为蛋白质如何调节酶的功能提供有价值的见解。最近的努力，利用RNase P的底物选择性工程定制核酶用于基因治疗，深入了解酶的功能的分子基础显然是必不可少的。