Enzymology of RNA Processing Enzymes

核糖核酸酶P（RNase P）催化前体tRNA（pre-tRNA）的5'端成熟，形成 tRNA是合成蛋白质所必需的成分。来自细菌的RNase P由RNA组成， 在体外催化前体tRNA切割的亚基和体内活性所必需的蛋白质组分 并增强前体tRNA底物的结合。相比之下，真核生物中的RNase P含有一种RNA， 多个蛋白质亚基。我们建议使用一种新的方法来研究细菌RNase P的功能。 生物化学和结构技术的结合。具体而言，我们的目标是：（1）探索结构和 使用时间分辨荧光共振能量转移技术测量RNase P的动力学 距离和迁移率;（2）研究细菌RNas P中的底物识别， RNase P中PRNA-pre-tRNA和pre-tRNA-P蛋白质接触的热力学和功能， 研究新的RNA底物的切割;（3）研究金属的位置和功能 与RNase P结合用于催化和底物识别;（4）描绘金属离子的位置 结合位点的RNase P和结构的分离螺旋NMR光谱分析。 我们的长期目标是进一步了解（1）核酶的催化机制， 蛋白酶，以及（2）RNA结合蛋白和蛋白质/RNA的结构和能量学 配合物RNase P是研究催化策略和底物识别的独特酶， 活性位点靠近蛋白质-RNA界面。这种蛋白质和RNA之间的独特协作 亚基可以提供深入了解从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。RNase P具有 作为一种新的抗生素靶点的潜在医学应用，因为它是一种必需的酶， 而原核生物的酶具有不同的亚基组成。提出的结构和功能研究 这里应该提供深入了解细菌核糖核酸酶P的活性位点定向抑制剂的发展， 目标生物如S.金黄色葡萄球菌和炭疽杆菌。

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 proposeto 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 an 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 andRNA subunits may provide insight into the evolution from RNAto 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 Pfrom target organisms such as S. aureus and Bacillus anthracis.