Enzymology of RNA Processing Enzymes

描述(申请人提供)：核糖核酸酶P(RNaseP)催化前体tRNA(前-tRNA)的5‘端成熟形成tRNA，这是蛋白质合成所必需的成分。细菌RNaseP由一个在体外催化Pre-tRNA裂解的RNA亚基和一个对体内活性至关重要并增强Pre-tRNA底物结合的蛋白质成分组成。相反，真核生物中的核糖核酸酶P含有一个RNA和多个蛋白质亚基。我们建议使用生化和结构技术相结合的方法来研究细菌RNaseP的功能。具体来说，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RNaseP的结构和动力学；(2)通过测定RNaseP中PRNA-pre-tRNA和Pre-tRNA-P蛋白质接触的热力学和功能以及研究新的RNA底物的切割，研究细菌RNAs P中的底物识别；(3)研究与RNaseP结合的金属在催化和底物识别方面的位置和功能；以及(4)通过核磁共振光谱分析确定RNaseP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)核酶相对于蛋白质酶的催化机制，以及(2)与RNA结合的蛋白质和蛋白质/RNA复合体的结构和能量。RNaseP是一种独特的研究催化策略和底物识别的酶，因为它的活性部位靠近蛋白质-RNA界面。蛋白质和RNA亚基之间的这种独特的合作可能为我们提供从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。核糖核酸酶P是一种重要的酶，且真核和原核酶具有不同的亚基组成，因此作为一种新型的抗生素靶标具有潜在的医疗应用价值。这里提出的结构和功能研究应该为从金黄色葡萄球菌和炭疽芽孢杆菌等靶生物中开发活性位点定向的细菌核糖核酸酶P抑制剂提供洞察力。

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.