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.
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