Structure and Function in Catalytic RNP Assembly

描述（由申请人提供）：本提案的广泛，长期目标是获得蛋白质如何在催化核糖核蛋白（RNP）复合体中调节RNA结构和功能的结构和动态见解。核糖核酸酶P （RNase P）是一种重要的、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'先导序列。真细菌酶是由一个RNA和一个蛋白质亚基组成的RNP复合体。RNA亚基本身具有催化作用，但蛋白质在体内是必需的。相比之下，人类的酶是由几个功能未知的蛋白质亚基组成的，其RNA成分本身似乎是无活性的。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是通过蛋白质和RNA亚基的分层相互结合诱导折叠进行的。使用来自嗜热古细菌的RNase P酶作为模型，研究人员将使用生化和生物物理工具来获得单个蛋白质成分（自由和复杂）与催化RNA亚基之间相互作用的结构见解。其目的是：1。通过核磁共振确定一个或多个蛋白质亚基（或其片段）的溶液结构。2. 利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（诱导配合）是否伴随RNP组装。这些目标的成功完成将使数据整合到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。随着最近利用RNase P的底物选择性来设计用于基因治疗的定制核酶的努力，深入了解酶功能的分子基础显然是必不可少的。

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.