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) 是一种 核糖核蛋白复合物，催化 5' 必需的成熟 前体 tRNA（前 tRNA）。蛋白质和 RNA 亚基对于 iii 体内活性；然而，细菌 RNA 成分催化前 tRNA 在高盐条件下进行体外裂解 iii。机械和结构研究 这种酶至关重要，因为 RNase P 和核糖体是唯一能发挥作用的核酶。 在体内发挥真正酶的作用。在上一个资助期内，我们解决了 核糖核酸酶 P 的蛋白质成分的第一个结构并被证明 该蛋白质通过结合前导序列来提高催化效率 前体-tRNA 底物。该功能将蛋白质成分 靠近活性位点，并首次证明 RNA 和 蛋白质成分都有助于分子识别特性。我们 建议继续研究其结构功能特性 使用生化组合的枯草芽孢杆菌 RNase P 蛋白 （诱变、交联、动力学分析、同位素效应、修饰 干涉和光谱）和 X 射线晶体结构 技术。具体来说，我们的目标是：（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) 划定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-寡核苷酸复合物的结构和 P 蛋白的位点特异性变体，并解析天然 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）机制 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/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 亚基之间的独特协作可能提供见解 从RNA到蛋白质催化剂的进化。最后，RNase P 作为新型抗生素靶标的潜在医学应用，因为它是 一种重要的原核酶，并作为特异性切割的新工具 基因治疗应用中的体内 mRNA 种类。