Molecular Dissection of Debranching Enzyme

脱支酶的分子解剖

• 批准号: 8848405
• 负责人: Subha Ranjan Das
• 金额: $ 27.95万
• 依托单位: CARNEGIE-MELLON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-06-01 至 2018-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8848405
• 关键词: Accounting; Active Sites; Address; Adenosine; Affect; Am 80; Binding; Biochemical; Biogenesis; Biological; Biological Assay; Boxing; Bypass; Calcineurin; Catalysis; Cell physiology; Cleaved cell; Complex; DNA Repair; Dissection; Dyes; Enzymes; Fluorescence; Future; Generations; Goals; HIV; Health; Heterogeneity; Human; Indium; Intervention; Introns; Kinetics; Label; Link; Manganese; Methods; MicroRNAs; Molecular; Molecular Conformation; Mus; Pathway interactions; Play; Positioning Attribute; Process; Property; Protein phosphatase; Proteins; RNA; RNA Binding; RNA Sequences; RNA Splicing; Reaction; Regulatory Pathway; Research; Retrotransposition; Roentgen Rays; Role; S Phase; Shapes; Solid; Solutions; Specificity; Spectrum Analysis; Structure; Testing; Therapeutic; Therapeutic Intervention; Trans-Splicing; Yeasts; analog; base; design; functional group; inhibitor/antagonist; insight; lariat debranching enzyme; mRNA Precursor; metalloenzyme; mutant; novel therapeutic intervention; nuclease; phosphodiester; phosphoric diester hydrolase; public health relevance; single molecule; transcriptome sequencing

DESCRIPTION (provided by applicant): The proposed research will provide new and comprehensive insight into debranching enzyme (Dbr1) that is a metalloenzyme central to the processing and biogenesis of regulatory RNAs. The use of modified branched RNA (bRNA) substrates and mimics will allow biochemical, structural and single-molecule studies into the binding, recognition and catalytic cleavage mechanism of Dbr1. This dissection of the molecular underpinnings of Dbr structure, function and dynamics is critical to the understanding of the unique specificity of Dbr1 and its active-site in cleavage of the bRNA 2'-5'-phophodiester cleaved. Through unprecedented access to synthetic bRNA, biochemical studies are proposed here to evaluate the active site residues and substrate requirements of Dbr1. Crystallographic analysis of Dbr1 will inform and enhance these biochemical studies by providing molecular details of protein and RNA contacts relevant to recognition, specificity and cleavage. Further, single-molecule spectroscopy will probe the dynamics of the macromolecular interactions to furnish important details into the reaction pathway and mechanism. The trifecta of methods proposed will provide a synergistic and cohesive account of the molecular details of Dbr1 cleavage of bRNA. These molecular details are critical for interventions through designed inhibitors in future therapeutic paradigms.

描述（由申请人提供）：拟议的研究将提供新的和全面的见解去分支酶（Dbr 1），这是一种金属酶的核心调控RNA的加工和生物发生。使用修饰的分支RNA（bRNA）底物和模拟物将允许对Dbr 1的结合，识别和催化切割机制进行生物化学，结构和单分子研究。Dbr结构、功能和动力学的分子基础的解剖对于理解Dbr 1及其在切割bRNA 2 '-5'-磷酸二酯中的活性位点的独特特异性是至关重要的。通过前所未有的合成bRNA，生化研究提出了在这里评估的活性位点残基和底物的需求Dbr 1。Dbr 1的晶体学分析将通过提供与识别、特异性和切割相关的蛋白质和RNA接触的分子细节来告知和增强这些生化研究。此外，单分子光谱将探测大分子相互作用的动力学，为反应途径和机制提供重要的细节。所提出的三种方法将提供Dbr 1切割bRNA的分子细节的协同和内聚性说明。这些分子细节对于在未来的治疗范例中通过设计的抑制剂进行干预至关重要。