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成分。来自细菌的RNase P由一个在体外催化前trna裂解的RNA亚基和一个对体内活性和增强前trna底物结合至关重要的蛋白质成分组成。而真核生物的RNase P含有一个RNA和多个蛋白亚基。我们建议使用生物化学和结构技术相结合的方法来研究细菌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来测量RNase P的距离和迁移率，探索RNase P的结构和动力学；(2)通过测定RNase P中PRNA-pre-tRNA和pre-tRNA-P蛋白接触的热力学和功能，以及研究新RNA底物的切割，研究细菌RNA P中的底物识别；(3)研究与RNase P结合的金属在催化和底物识别中的位置和功能；(4)通过核磁共振光谱分析确定了RNase P中金属离子结合位点的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是一种独特的酶，用于研究催化策略和底物识别，因为其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合作可能为从RNA到蛋白质催化剂的进化提供洞见。RNase P是一种必需的酶，因为tRNA成熟是蛋白质合成所必需的。由于RNase P是一种必需酶，并且真核和原核酶具有不同的亚基组成，因此它作为一种新的抗生素靶点具有潜在的医学应用价值。本文提出的结构和功能研究将为从目标生物（如金黄色葡萄球菌和炭疽芽孢杆菌）开发细菌RNase P的活性位点定向抑制剂提供见解。

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.