Enzymology of RNA Processing Enzymes

描述（由申请人提供）：核糖核酸酶P（RNase P）催化前体tRNA（pre-tRNA）的5'端成熟形成tRNA，tRNA是合成蛋白质所必需的组分。来自细菌的RNase P由在体外催化前体tRNA切割的RNA亚基和对于体内活性是必需的并且增强前体tRNA底物的结合的蛋白质组分组成。相比之下，真核生物中的RNase P包含一个RNA和多个蛋白质亚基。我们建议使用生物化学和结构技术相结合来研究细菌RNase P的功能。具体而言，我们的目标是：（1）利用时间分辨荧光共振能量转移技术测量距离和迁移率，探索RNase P的结构和动力学;（2）通过确定RNase P中PRNA-pre-tRNA和pre-tRNA-P蛋白接触的热力学和功能，研究细菌RNas P中的底物识别，并研究新型RNA底物的切割;（3）研究金属离子与RNase P结合的位置和功能，以及它们在RNase P中的催化和底物识别作用;（4）通过NMR光谱分析，确定金属离子在RNase P中的结合位置和分离螺旋的结构。我们的长期目标是进一步了解（1）与蛋白酶相比，核酶使用的催化机制，以及（2）RNA结合蛋白和蛋白质/RNA复合物的结构和能量学。RNase P是研究催化策略和底物识别的独特酶，因为活性位点靠近蛋白质-RNA界面。蛋白质和RNA亚基之间的这种独特的合作可能会提供深入了解从RNA到蛋白质催化剂的演变。RNase P是一种必需的酶，因为tRNA成熟是蛋白质合成所必需的。核糖核酸酶P是一种重要的酶，而且真核生物和原核生物的核糖核酸酶具有不同的亚基组成，因此核糖核酸酶P作为一种新型的抗生素靶标具有潜在的医学应用价值。本文提出的结构和功能研究应该为从靶生物如S.金黄色葡萄球菌和炭疽杆菌。

DESCRIPTION (provided by applicant): Ribonuclease P (RNase P) catalyzes the maturation of the 5' end of precursor tRNA (pre-tRNA) to form tRNA, a component essential for the synthesis of proteins. RNase P from bacteria is composed of an RNA subunit that catalyzes pre-tRNA cleavage in vitro and a protein component that is essential for activity in vivo and enhances binding of the pre-tRNA substrate. In contrast, RNase P in eukaryotes contains one RNA and multiple protein subunits. We propose to investigate the function of the bacterial RNase P using a combination of biochemical and structural techniques. Specifically, we aim to: (1) explore the structure and dynamics of RNase P using time resolved fluorescence resonance energy transfer techniques to measure distances and mobility; (2) investigate substrate recognition in bacterial RNas P by determining the thermodynamics and function of PRNA-pre-tRNA and pre-tRNA-P protein contacts in RNase P and investigating the cleavage of novel RNA substrates; (3) investigate the position and functions of metals bound to RNase P for both catalysis and substrate recognition; and (4) delineate the position of metal ion binding sites in RNase P and the structure of isolated helices by NMR spectroscopic analysis. Our long term goal is to further understand (1) the mechanisms of catalysis used by ribozymes as compared to protein enzymes, and (2) the structure and energetics of RNA binding proteins and protein/RNA complexes. RNase P is a unique enzyme to investigate catalytic strategies and substrate recognition since the active site is near the protein-RNA interface. This unique collaboration between the protein and RNA subunits may provide insight into the evolution from RNA to protein catalysts. RNase P is an essential enzyme as tRNA maturation is required for protein synthesis. RNase P has potential medical applications as a novel antibiotic target since it is an essential enzyme and the eukaryotic and prokaryotic enzymes have different subunit composition. The structural and functional studies proposed here should provide insight into the development of active site-directed inhibitors of bacterial RNase P from target organisms such as S. aureus and Bacillus anthracis.