The long-term goal is to understand the structure and enzymatic mechanism of small catalytic RNAs (ribozymes). Current efforts are focused on the hammerhead and hepatitis delta virus ribozymes. Small ribozymes are being used to attenuate proliferation of viruses and specific mammalian cell types; knowledge of their structure will aid their development as stable, effective therapeutic agents. The x-ray crystallographic structure of the hammerhead ribozyme has been solved, revealing the overall tertiary fold of the molecule. However, the ribozyme is in an apparent kinetic ground state , the structure does not reveal the catalytic mechanism. Further studies of structure and actuvity will be directed toward stabilizing ribozyme-inhibitor complexes that mimic intermediates approaching the transition state of the reaction. This work will use a spectrum of inhibitors with modifications around the active site, as well as a spectrum of divalent ions to substitute for the catalytic metal ion. The work will proceed in three stages: I. Solve the structure of currently-available crystals to high (greater than or equal to 2.0 Angstrom units) resolution. II. Using crystals isomorphous to those of stage I when possthe structures of a spectrum of inhibitor each of which will have different interactions within the active site. Scan divalent ion binding at the active site with Mg2+, Cd2+ and Cr2+. Assay for activity in crystals and in solution. III. Using information from stage II, stabilize complexes between inhibitors/substrates and specific divalent ions that mimic reaction intermediate states and solve their structures to high resolution. When completed, this work should give and unambiguous picture of the structural basis for catalytic activity by the hammerhead ribozyme. Additionally, ongoing efforts to improve the quality of currently- available crystals of the hepatitis delta virus ribozyme will be continued, with the goal of solving the ribozyme structure. This ribozyme catalyzes the same reaction as the hammerhead but utilizes a completely different tertiary structure for this activity; its structure will provide new insights on ribozyme mechanism.
| Wedekind, Joseph E; McKay, David B (2003) Crystal structure of the leadzyme at 1.8 A resolution: metal ion binding and the implications for catalytic mechanism and allo site ion regulation. Biochemistry 42:9554-63 |
| Wedekind, J E; McKay, D B (1998) Crystallographic structures of the hammerhead ribozyme: relationship to ribozyme folding and catalysis. Annu Rev Biophys Biomol Struct 27:475-502 |
| McKay, D B (1996) Structure and function of the hammerhead ribozyme: an unfinished story. RNA 2:395-403 |
| Pley, H W; Lindes, D S; DeLuca-Flaherty, C et al. (1993) Crystals of a hammerhead ribozyme. J Biol Chem 268:19656-8 |
