Investigation of the Hairpin Ribozyme Tertiary Structure

描述(由申请人提供)：本提案的目的是了解 发夹状核酶催化的结构基础，从而 建立基于RNA的新型核糖核酸的长期发展框架 治疗学。这种发夹在小核酶家族的成员中是独一无二的， 因为反应的内部平衡有利于连接而不是切割， 金属离子不直接参与化学步骤。 反应。发夹次级折叠以保守的核心为特征 由两个内部环域组成，其第三级相互作用是关键的 合成具有活性的折叠酶。通过求解三维结构 对于发夹核酶，韦德金德博士将能够识别功能 在域间交错处进行第三级联系的组，以及 催化所需的特定立体化学约束。具体目标 主要有：(I)求出一个结构的最小核苷酸构造量 发夹状核酶。晶体将X射线衍射到标称的3.3A分辨率， 属于晶胞尺寸为a=94.0A的空间群P61 22(或P65 22 和c=123.0 A。这一目标将揭示核糖核酸酶的整体折叠； 为了解决含有适量底物的复合体中的发夹结构- 和产品类似物。这些结构将揭示 酶活性部位潜在的酸/碱催化剂；(Iii)测量 各自天然与非天然的结构域间平衡解离常数 表面等离子体共振突变的发夹状核酶。使用 结构作为指南，韦德金德博士将记录和对比 各种离子，以及域间界面上的单原子取代。 这一方法学的发展将为证实HIS提供基础 使用模拟条件的溶液测量进行结构观测 结晶的过程。发夹反应的详细催化机理 然后将被建造。从长远来看，全面理解 核酶将是构建新的基因治疗试剂和 模仿RNA的基本结构和化学结构的药物 酵素。

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.