Structure and Function in Catalytic RNP Assembly

催化 RNP 组装的结构和功能

基本信息

批准号：
7936606
负责人：
MARK P. FOSTER
金额：
$ 25.16万
依托单位：
OHIO STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-09-30 至 2011-03-31
项目状态：
已结题

项目摘要

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.

描述（由申请人提供）：该建议的广泛，长期目标是获得结构和动态见解，以了解蛋白质如何调节催化核糖核蛋白（RNP）复合物中的RNA结构和功能。核糖核酸酶P（RNase P）是一种必不可少的核糖核蛋白酶，主要负责在TRNA成熟过程中裂解5'领导者序列。胚细菌酶是由一个RNA和一个蛋白亚基组成的RNP复合物。 RNA亚基本身是催化性的，但是在体内需要蛋白质。相反，具有未知功能的几个蛋白质亚基构成人类酶，其RNA成分似乎是不活跃的。研究人员建议，蛋白质亚基的主要作用是稳定RNA的主动构象，全酶组装和底物识别通过层次相互结合诱导的蛋白质和RNA亚基进行。研究人员将使用来自嗜热古细菌的RNase P酶作为模型，将使用生化和生物物理工具来获得与催化RNA亚基的自由和复杂的单个蛋白质成分之间的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。表征天然和体外重构酶中蛋白质 - 蛋白质和蛋白-RNA相互作用，并检查RNP组装是否伴随着构象变化（诱导拟合）。这些目标的成功完成将使数据能够将数据集成到RNase P Holoenzyme的三维模型中，并为蛋白质如何调节酶的功能提供宝贵的见解。在最近努力利用RNase P的底物选择性来设计用于基因治疗的定制核酶的底物选择性，对酶功能的分子基础的见解显然至关重要。