Molecular Dissection of Debranching Enzyme

描述(由申请人提供)：拟议的研究将提供对脱支酶(DBR1)的新的和全面的见解，DBR1是一种对调控RNA的加工和生物发生至关重要的金属酶。使用修饰的分支RNA(Brna)底物和模拟物将允许对DBR1的结合、识别和催化切割机制进行生化、结构和单分子研究。这种对DBR结构、功能和动力学的分子基础的剖析对于理解DBR1的独特专一性及其在裂解Brna 2‘-5’-磷酸二酯中的活性部位是至关重要的。通过史无前例地获得合成的Brna，这里提出了评估DBR1的活性部位残基和底物需求的生化研究。DBR1的结晶学分析将提供与识别、特异性和切割相关的蛋白质和RNA接触的分子细节，从而为这些生化研究提供信息并加强这些研究。此外，单分子光谱学将探索大分子相互作用的动力学，为了解反应途径和机理提供重要细节。所提出的三种方法将提供对Brna的DBR1裂解的分子细节的协同和内聚的描述。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。

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.