Molecular Dissection of Debranching Enzyme

脱支酶的分子解剖

• 批准号: 8673376
• 负责人: Subha Ranjan Das
• 金额: $ 27.87万
• 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/8673376
• 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

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）：拟议的研究将为脱粒酶（DBR1）提供新的全面见解，这是对调节RNA的加工和生物发生的中心的金属酶。修饰的分支RNA（BRNA）底物和模拟物的使用将使DBR1的结合，识别和催化裂解机理允许生化，结构和单分子研究。 DBR结构，功能和动力学的分子基础的这种解剖对于理解DBR1的独特特异性及其在BRNA 2'-5'-5'-Phophophophodiester裂解时的主动位置至关重要。通过前所未有的访问合成BRNA，这里提出了生化研究，以评估DBR1的活性位点残基和底物需求。 DBR1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接触的分子细节来为这些生化研究提供信息和增强。此外，单分子光谱法将探测大分子相互作用的动力学，以将重要细节提供到反应途径和机理中。提出的方法的三fecta将提供有关BRNA DBR1裂解的分子细节的协同和内凝性。这些分子细节对于通过未来的治疗范式中设计的抑制剂进行干预至关重要。