ENZYMOLOGY OF RNA PROCESSING ENZYMES

描述(申请人描述)：核糖核酸酶P(RNaseP)是一种 催化细胞基本5‘成熟的核糖核蛋白复合体 前体tRNA(前tRNA)。蛋白质和核糖核糖核酸亚基都是 活体活性；然而，细菌RNA组分催化前-tRNA 在高盐中进行III体外卵裂。机械和结构方面的研究 这种酶是至关重要的，因为RNaseP和核糖体是唯一能够 在体内发挥真正的酶的作用。在上一个资助期，我们解决了 核糖核酸酶P蛋白组分的一级结构及证明 蛋白质通过结合前导序列来提高催化效率 前体-tRNA底物。该函数将蛋白质组分 并首次证明了RNA和 蛋白质组分都对分子识别特性有贡献。我们 建议继续研究化合物的结构-功能特性 利用枯草芽孢杆菌核糖核酸酶P蛋白的组合生化 (诱变、交联、动力学分析、同位素效应、修饰 干涉和光谱学)和x射线晶体结构 技巧。具体来说，我们的目标是：(1)探索分子识别 核糖核酸酶P蛋白中单链RNA结合裂隙的性质和TO 确定该位置相对于P RNA的位置；(2)描绘P 蛋白质/P RNA结合表面并定义特定接触在其中的作用 催化；(3)确定催化金属中心的位置并解剖 催化机理；以及(4)延长RNaseP蛋白的分辨率 结构，确定P蛋白-寡核苷酸复合体的结构和 P蛋白的定点变异，并解决了天然RNaseP的结构 全酶和全酶-底物复合体。 我们的长期目标是加深我们对(1) 与蛋白质酶相比，核酶所用的催化作用，以及(2) RNA结合蛋白和蛋白质/RNA复合体的结构和能量学。 核糖核酸酶P是比较蛋白质和RNA中催化策略的理想酶 酶，因为它在体内的生物合成途径中发挥作用。此外， 蛋白质和RNA亚基之间的独特合作可能提供洞察力 从RNA到蛋白质催化剂的进化。最后，RNaseP有 潜在的医疗应用既是一种新的抗生素靶标，因为它是 一种必需的原核酶，并作为一种新的工具来特异性切割 基因在体内的物种在基因治疗中的应用。

DESCRIPTION (applicant's description): Ribonuclease P (RNase P) is a ribonucleoprotein complex that catalyzes the essential 5' maturation of precursor tRNA (pre-tRNA). Both the protein and RNA subunits are essential for iii vivo activity; however, the bacterial RNA component catalyzes pre-tRNA cleavage iii vitro in high salt. Mechanistic and structural investigations of this enzyme are crucial as RNase P and the ribosome are the only ribozymes that function as true enzymes in vivo. In the previous grant period, we solved the first structure of the protein component of ribonuclease P and demonstrated that the protein enhances catalytic efficiency by binding the leader sequence of the precursor-tRNA substrate. This function places the protein component near the active site and for the first time demonstrates that the RNA and protein components both contribute to molecular recognition properties. We propose to continue investigating the structure-function properties of the Bacillus subtilis RNase P protein using a combination of biochemical (mutagenesis, crosslinking, kinetic analysis, isotope effects, modification interference, and spectroscopy) and x-ray crystallographic structural techniques. Specifically, we aim to: (1) explore the molecular recognition properties of the single-strand RNA binding cleft in the RNase P protein and to determine the position of this site relative to the P RNA; (2) delineate the P protein/P RNA binding surface and define the role of specific contacts in catalysis; (3) determine the position of catalytic metal sites and dissect the catalytic mechanism; and (4) extend the resolution of the RNase P protein structure, determine the structures of P protein-oligonucleotide complexes and site-specific variants of P protein, and solve the structure of native RNase P holoenzyme and holoenzyme-substrate complexes. Our long-term goal is to further our understanding of (1) the mechanisms of catalysis used by ribozymes as compared to protein enzymes, and (2) the structures and energetics of RNA binding proteins and protein/RNA complexes. RNase P is an ideal enzyme to compare catalytic strategies in protein and RNA enzymes since it functions in a biosynthetic pathway in vivo. Furthermore, the unique collaboration between the protein and RNA subunits may provide insight into the evolution from RNA to protein catalysts. Finally, RNase P has potential medical applications as both a novel antibiotic target, since it is an essential prokaryotic enzyme, and as a novel tool to specifically cleave mRNA species in vivo in gene therapy applications.