Structure of a ribozyme catalytic center

核酶催化中心的结构

• 批准号: 7630419
• 负责人: KWAKU DAYIE
• 金额: $ 27.68万
• 依托单位: UNIV OF MARYLAND, COLLEGE PARK
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-08-01 至 2012-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7630419
• 关键词: Active Sites; Affinity; Binding; Biochemical; Biochemical Genetics; Biochemistry; Biological Assay; Biological Models; Catalysis; Catalytic Domain; Catalytic RNA; Cells; Chemistry; Cleaved cell; Complex; Coupling; Data; Docking; Elements; Employee Strikes; Enzymes; Eukaryotic Cell; Exons; Fluorescence; Foundations; Fred Hutchinson Cancer Research Center; Gel; Genes; Goals; Introns; Ions; Knowledge; Label; Learning; Maps; Metal Binding Site; Methods; Modeling; Molecular; Molecular Conformation; Molecular Machines; Monitor; Mutagenesis; NMR Spectroscopy; Nucleotides; Phylogenetic Analysis; Physiological; Proteins; RNA; RNA Splicing; Reaction; Research; Research Personnel; Residual state; Resolution; Role; Side; Site-Directed Mutagenesis; Small Nuclear RNA; Sodium Chloride; Solutions; Spliceosomes; Structure; Study models; Substrate Domain; System; Techniques; Therapeutic; Time; Transcript; UV Mutagenesis; Work; X-Ray Crystallography; base; crosslink; domain mapping; functional group; hammerhead ribozyme; insight; mutant; novel; novel strategies; nucleotide analog; programs; stable isotope; stem; three dimensional structure; tool

DESCRIPTION (provided by applicant): For most genes in eukaryotic cells, splicing of the primary transcript is undertaken by a megadalton molecular machine, the spliceosome, consisting of over 100 proteins and five small nuclear RNAs (snRNAs). However, this splicing reaction is also carried out by the Group II intron RNA enzymes (ribozymes) without the need of proteins. Such ribozymes share striking structural and mechanistic similarities to the spliceosome, which makes them an attractive model for studying the basic biochemistry of splicing. It is now well established that domains 1 through 3 (D123), domain 5 (D5) and exon substrates form the minimal active site essential for catalysis by group II introns. The only known structure of the active site component is D5 in isolation. Yet D5 alone cannot mimic the active catalytic configuration. Examining the structure of D5 in the context of D123 and substrates is therefore critical for advancing our knowledge of atomic basis for catalysis. To this end we have reconstructed a group II intron from Pylaiella littoralis into a novel tripartite ribozyme system that has some important difference from previous model systems and that makes it an attractive target for structural studies. We hypothesize that the internal bulge of D5 and substrate form a key part of the catalytic site. We will apply powerful biophysical and biochemical tools to obtain detailed structural insight into the minimal catalytic core of this ribozyme. Our overall specific aims are: 1. Determine the molecular basis for the catalytic deficiency of a D5 bulge deletion mutant-D5A25delta. 2. Map the binding interface between D5 bulge mutant and D123 in the presence and absence of substrates using NMR, mutagenesis, and UV crosslinking 3. To obtain an atomic view of the minimal active site and to identify the features that are important for binding and catalysis, determine the structure of D5 and D5 bulge mutant bound to D123 (and or fragments of D123) and substrate using NMR, and 4. Validate the structure using biochemical assays. Significance: We anticipate that completion of these studies will provide an atomic framework for interpreting the extensive biochemical and genetic data on group II introns to extend our understanding of catalysis, these might provide the first evidence for the details of RNA interactions in cell splicing, and suggest novel approaches to developing group II introns as potential ribozyme therapeutics.

描述（由申请人提供）：对于真核细胞中的大多数基因，初级转录本的剪接是由兆道尔顿分子机器（剪接体）进行的，剪接体由 100 多种蛋白质和 5 个小核 RNA (snRNA) 组成。然而，这种剪接反应也是由 II 组内含子 RNA 酶（核酶）完成的，而不需要蛋白质。这种核酶与剪接体具有惊人的结构和机制相似性，这使得它们成为研究剪接的基本生物化学的有吸引力的模型。现在已经确定，结构域 1 至结构域 3 (D123)、结构域 5 (D5) 和外显子底物形成了 II 组内含子催化所必需的最小活性位点。唯一已知的活性位点成分结构是孤立的 D5。然而，单独的 D5 无法模拟活性催化构型。因此，在 D123 和底物的背景下检查 D5 的结构对于增进我们对催化原子基础的了解至关重要。为此，我们将来自Pylaiella littoralis的II组内含子重建为新型三联核酶系统，该系统与以前的模型系统有一些重要的区别，这使其成为结构研究的有吸引力的目标。我们假设 D5 和底物的内部凸起形成催化位点的关键部分。我们将应用强大的生物物理和生化工具来深入了解这种核酶的最小催化核心的详细结构。我们总体的具体目标是： 1.确定D5突出缺失突变体-D5A25delta催化缺陷的分子基础。 2. 使用 NMR、诱变和 UV 交联绘制在存在和不存在底物的情况下 D5 凸出突变体和 D123 之间的结合界面。 3. 为了获得最小活性位点的原子视图并识别对结合和催化重要的特征，使用 NMR 确定与 D123（和/或 D123 片段）和底物结合的 D5 和 D5 凸出突变体的结构， 4. 使用生化分析验证结构。意义：我们预计这些研究的完成将为解释第二组内含子的广泛生化和遗传数据提供一个原子框架，以扩展我们对催化的理解，这些可能为细胞剪接中RNA相互作用的细节提供第一个证据，并提出开发第二组内含子作为潜在核酶疗法的新方法。