ENZYMOLOGY OF RNA PROCESSING ENZYMES

描述（申请人的描述）：核糖核酸酶P（RNase P）是一种 一种核糖核蛋白复合物，催化 前体tRNA（pre-tRNA）。蛋白质和RNA亚基都是必需的， iii体内活性;然而，细菌RNA组分催化前tRNA 在高盐中体外切割III。机械和结构调查 这种酶是至关重要的核糖核酸酶P和核糖体是唯一的核酶， 在体内发挥真正的酶的作用。在上一个赠款期间，我们解决了 核糖核酸酶P的蛋白质组分的第一个结构，并证明 该蛋白质通过结合前导序列增强催化效率， 转移核糖核酸的底物。这个功能将蛋白质成分 在活性位点附近，首次证明了RNA和 蛋白质组分都有助于分子识别特性。我们 建议继续研究的结构功能特性， 枯草芽孢杆菌RNase P蛋白使用生物化学的组合 （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 技术.具体而言，我们的目标是：（1）探索分子识别 RNA酶P蛋白中单链RNA结合裂缝的性质， 确定该位点相对于P RNA的位置;（2）描绘P RNA的位置。 蛋白质/P RNA结合表面，并定义了特异性接触在 催化;（3）确定催化金属位点的位置，并解剖 催化机制;（4）延长RNase P蛋白的分辨率 结构，确定P蛋白-寡核苷酸复合物的结构， P蛋白的位点特异性变体，并解决了天然RNase P的结构 全酶和全酶-底物复合物。 我们的长期目标是进一步了解（1） 与蛋白酶相比，核酶使用的催化作用，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是比较蛋白质和RNA催化策略的理想酶 酶，因为它在体内的生物合成途径中起作用。而且 蛋白质和RNA亚基之间的独特协作可以提供洞察力， 从RNA到蛋白质催化剂的进化。最后，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 一种必需的原核酶，并作为一种新的工具， mRNA在体内基因治疗中的应用。

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.