Structure and Function in Catalytic RNP Assembly

描述(由申请人提供)：这项建议的广泛、长期目标是获得对蛋白质如何调节催化核糖核蛋白(RNP)复合体中的RNA结构和功能的结构和动态的见解。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，在tRNAs成熟过程中主要负责切割5‘前导序列。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亚单位本身是催化的，但蛋白质在体内是必需的。相比之下，几个功能未知的蛋白质亚基构成了人类的酶，其RNA成分单独似乎是不活跃的。研究人员认为，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。以一种嗜热古细菌的RNaseP酶为模型，研究人员将使用生化和生物物理工具来获得对单个蛋白质组分之间相互作用的结构性见解，无论是游离的还是复杂的，与催化的RNA亚基。其目的是：1.用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.鉴定天然和体外重组酶中蛋白质-蛋白质和蛋白质-RNA的相互作用，并检测构象变化(诱导FIT)是否伴随着RNP组装。这些目标的成功完成将使数据能够整合到RNaseP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RNaseP的底物选择性来设计用于基因治疗的定制核酶的努力，对该酶功能的分子基础的深入了解显然是必不可少的。

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.