Enzymology of RNA Processing Enzymes

核糖核酸酶P(RNaseP)催化前体tRNA(Pre-tRNA)5‘端成熟形成 TRNA，蛋白质合成所必需的成分。细菌的核糖核酸酶P由一个RNA组成 体外催化Pre-tRNA裂解的亚基和体内活性所必需的蛋白质成分 并增强与前tRNA底物的结合。相比之下，真核生物中的RNaseP只有一个RNA和 多个蛋白质亚基。我们建议用一种新的方法研究细菌核糖核酸酶P的功能。 生化技术和结构技术的结合。具体地说，我们的目标是：(1)探索结构和 时间分辨荧光共振能量转移技术测定核糖核酸酶P的动力学 距离和迁移率；(2)通过确定细菌RNA P的底物识别 RNaseP和RNaseP中PRNA-Pre-tRNA和Pre-tRNA-P蛋白接触的热力学和功能 研究新的RNA底物的裂解；(3)研究金属的位置和作用 与核糖核酸酶P结合，用于催化和底物识别；以及(4)描绘金属离子的位置 核磁共振光谱分析RNaseP中的结合部位和分离螺旋的结构。 我们的长期目标是通过比较进一步了解(1)核酶的催化作用机制 (2)RNA结合蛋白和蛋白质/RNA的结构和能量学 复合体。核糖核酸酶P是一种独特的酶，用于研究催化策略和底物识别 活性部位位于蛋白质-RNA界面附近。蛋白质和RNA之间的这种独特的合作 亚基可能提供从RNA到蛋白质催化剂的进化的洞察力。 RNaseP是一种必需的酶，因为蛋白质合成需要tRNA的成熟。RNaseP有 由于它是一种必需的酶和真核生物，因此作为一种新的抗生素靶点具有潜在的医学应用 原核酶具有不同的亚基组成。建议的结构和功能研究 这将为细菌核糖核酸酶活性位点定向抑制剂的发展提供洞察力 目标生物，如金黄色葡萄球菌和炭疽芽孢杆菌。

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.