Structure and Function in Catalytic RNP Assembly

描述（由申请人提供）：该提案的广泛、长期目标是获得关于蛋白质如何调节催化核糖核蛋白 (RNP) 复合物中 RNA 结构和功能的结构和动态见解。核糖核酸酶 P (RNase P) 是一种必需且普遍存在的核糖核蛋白酶，主要负责在 tRNA 成熟过程中切割 5' 前导序列。真细菌酶是一种 RNP 复合物，由一种 RNA 和一种蛋白质亚基组成。 RNA亚基本身具有催化作用，但体内需要蛋白质。相比之下，人类酶由几个功能未知的蛋白质亚基构成，而其单独的 RNA 成分似乎没有活性。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是通过蛋白质和 RNA 亚基的分层相互结合诱导折叠进行的。研究人员将使用来自嗜热古细菌的 RNase P 酶作为模型，利用生化和生物物理工具来深入了解单个蛋白质成分（游离的和复合的）与催化 RNA 亚基之间的相互作用。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. 使用酶探针和化学探针绘制 RNA 亚基的二级结构和蛋白质结合位点图。 3. 表征天然和体外重组酶中蛋白质-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。成功完成这些目标将使数据能够整合到 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，人们努力利用 RNase P 的底物选择性来设计用于基因治疗的定制核酶，因此深入了解该酶功能的分子基础显然至关重要。

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.