Molecular Dissection of Debranching Enzyme

描述（由申请人提供）：拟议的研究将为脱分枝酶（Dbr1）提供新的和全面的见解，Dbr1是一种金属酶，对调控rna的加工和生物发生至关重要。使用修饰的支链RNA （bRNA）底物和模拟物将允许对Dbr1的结合、识别和催化裂解机制进行生化、结构和单分子研究。对Dbr结构、功能和动力学的分子基础的解剖对于理解Dbr1的独特特异性及其在bRNA 2'-5'-phophodiester裂解中的活性位点至关重要。通过前所未有地获得合成bRNA，本文提出了生化研究来评估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，为这些生化研究提供信息和增强。此外，单分子光谱学将探索大分子相互作用的动力学，为反应途径和机理提供重要的细节。提出的三种方法将为Dbr1切割bRNA的分子细节提供协同和内聚的解释。这些分子细节对于在未来的治疗范例中通过设计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.