ENZYMOLOGY OF RNA PROCESSING ENZYMES

RNA 加工酶的酶学

• 批准号: 6627210
• 负责人: CAROL A FIERKE
• 金额: $ 45.42万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-01-01 至 2004-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6627210
• 关键词: X ray crystallography; active sites; enzyme activity; enzyme biosynthesis; enzyme mechanism; enzyme structure; enzyme substrate analog; fluorescence resonance energy transfer; ribonuclease P; ribozymes; thiophosphate; transfer RNA

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.

描述（申请人的描述）：核糖核酸酶P（RNase P）是一个 核糖核蛋白复合物催化必需的5'成熟 前体tRNA（pre-tRNA）。蛋白质和RNA亚基对于 III体内活动；但是，细菌RNA成分催化了pre-tRNA 在高盐中的裂解III体外。机械和结构调查 这种酶至关重要，因为RNase P，核糖体是唯一的核酶 在体内充当真正的酶。在上一个赠款期间，我们解决了 核糖核酸酶P的蛋白质成分的第一结构，并证明 蛋白质通过结合领导者序列来提高催化效率 前体-TRNA底物的。此功能放置蛋白质成分 在活动地点附近，第一次证明了RNA和 蛋白质成分均有助于分子识别特性。我们 建议继续研究该结构功能 枯草芽孢杆菌RNase P蛋白使用生化的组合 （诱变，交联，动力学分析，同位素效应，修饰 干扰和光谱法）和X射线晶体学结构 技术。具体而言，我们的目的是：（1）探索分子识别 RNase P蛋白中单链RNA结合裂缝的特性 确定该位点相对于P RNA的位置； （2）描述P 蛋白质/P RNA结合表面并定义特定接触的作用 催化; （3）确定催化金属位点的位置并剖析 催化机制； （4）扩展RNase P蛋白的分辨率 结构，确定P蛋白 - 寡核苷酸络合物的结构和 P蛋白的位点特异性变体，并求解天然RNase P的结构 Holoenzyme和Holoenzyme-Substrate复合物。 我们的长期目标是进一步了解（1） 与蛋白质酶相比，核酶使用的催化作用，（2） RNA结合蛋白和蛋白/RNA复合物的结构和能量。 RNase P是比较蛋白质和RNA中催化策略的理想酶 酶在体内的生物合成途径中起作用。此外， 蛋白质和RNA亚基之间的独特合作可能会提供洞察力 从RNA到蛋白质催化剂的演变。最后，RNase P具有 潜在的医疗应用是一种新型的抗生素靶标，因为它是 必需的原核酶，也是特定裂解的新工具 基因治疗应用中体内mRNA物种。