Investigation of the Hairpin Ribozyme Tertiary Structure

描述（由申请人提供）：本提案的目标是了解 发夹状核酶催化作用的结构基础， 建立一个框架，为长期发展新的基于RNA的 治疗学发夹结构在小核酶家族中是独一无二的， 因为反应的内部平衡有利于连接而不是裂解， 并且金属离子不直接参与反应的化学步骤。 反应发夹二级折叠的特征是一个保守的核心 由两个内环结构域组成，其三级相互作用至关重要 合成活性折叠酶。通过解决三维结构 Wedekind博士将能够识别出 在域间界面进行三级接触的团体，以及 催化所必需的特定立体化学约束。具体目标 （i）解决最小的64个核苷酸构建体的结构， 发夹状核酶。晶体将X射线反射到标称的3.3 A分辨率， 属于空间群P61 22（或P65 22），晶胞尺寸a = 94.0 c = 123.0A。这一目标将揭示RNA酶的整体折叠;（ii） 来解决发夹结构与适度数量的底物的复杂性- 和产品类似物。这些结构将揭示 酶活性位点中潜在的酸/碱催化剂;（iii）测量酶活性位点中潜在的酸/碱催化剂。 各自的天然与非天然的结构域间平衡解离常数 突变的发夹状核酶。使用 作为指导，Wedekind博士将记录和对比 各种离子和畴间界面处的单原子取代。 这种方法的发展将提供一个基础，以证实他的 使用模拟条件的溶液测量进行结构观测 结晶化。发夹反应的详细催化机理 将被建造。从长远来看，全面了解 核酶对于构建新的基因治疗剂是必不可少的， 模仿RNA的基本结构和化学性质的药物 内切酶

DESCRIPTION (provided by applicant): The goal of this proposal is to understand the structural basis for catalysis by the hairpin ribozyme, thereby establishing a framework for the long-term development of new RNA-based therapeutics. The hairpin is unique among members of the small ribozyme family, in that the internal equilibrium of the reaction favors ligation over cleavage, and metal ions do not participate directly in the chemical steps of the reaction. The hairpin secondary fold is characterized by a conserved core composed of two internal loop domains whose tertiary interactions are critical to compose the active folded enzyme. By solving the three-dimensional structure of the hairpin ribozyme, Dr. Wedekind will be able to identify functional groups engaged in tertiary contacts at the interdomain interlace, as well as specific stereochemical constraints necessary for catalysis. The specific aims are: (i) to solve the structure of a 64-nucleotide construct of the minimal hairpin ribozyme. Crystals diffract X-rays to a nominal 3.3 A resolution and belong to space group P61 22 (or P65 22) with unit cell dimensions a = 94.0 A and c = 123.0 A. This aim will reveal the overall fold of the RNA enzyme; (ii) to solve the hairpin structure in complex with a modest number of substrate- and product-analogs. These structures will reveal the spatial distribution of potential acid/base catalysts in the enzyme active site; (iii) to measure the interdomain equilibrium dissociation constants of respective native versus mutated hairpin ribozymes by means of surface plasmon resonance. Using the structure as a guide, Dr. Wedekind will record and contrast the effects of various ions, and single atom substitutions at the interdomain interface. Development of this methodology will provide a basis to corroborate his structural observations using solution measurements that mimic the conditions of crystallization. A detailed catalytic mechanism for the hairpin reaction will then be constructed. In the long term, a comprehensive understanding of ribozymes will be essential for the construction of new gene therapy agents and pharmaceuticals that mimic the fundamental architecture and chemistry of RNA enzymes.