Our work explores two interesting aspects of catalytic RNAs, or ribozymes. First, we investigate the mechanisms by which RNA molecules fold into complex three-dimensional structures and catalyze chemical reactions. Since the catalytically active structures of these molecules can be unstable, and the molecules are often susceptible to misfolding, it has taken an approach utilizing many different tools and techniques to discover how they carry out their reactions. In our studies of the hairpin ribozyme, a 50-nucleotide motif that catalyzes a reversible cleavage of a target RNA sequence, we re-engineered the ribozyme to minimize misfolding and then used a battery of techniques-fluorescence studies, reaction kinetics, backbone protection, photo-crosslinking and molecular modeling with the MC-SYM program-to characterize the active structure of the ribozyme-substrate complex. The resulting model implicated a particular guanosine residue in the ribozyme as a likely participant in chemical catalysis by the molecule, and further studies confirmed its importance in the cleavage reaction. We continue to use a combination of biochemistry and computer modeling in studying this ribozyme, and we are also applying these methods to a new study of the hammerhead ribozyme. The hammerhead is a small, much-studied RNA often used to cleave target RNAs in cells and animals. Despite the availability of a crystal structure, the catalytic conformation and mechanism of the hammerhead have not been elucidated. The approaches which have allowed us to probe the function of the hairpin will hopefully reveal the elusive mechanism of this important ribozyme.
Use of Hairpin Ribozymes as Antiviral Agents in Cells
Pinard, R., Hampel, K.J., Heckman, J.E., Lambert, D., Major, F., Burke, J.M. Functional involvement of G8 in the hairpin ribozyme cleavage mechanism. EMBO J. 2001, 20:6434-6442.
Pinard, R., Lambert, D., Heckman, J.E., Esteban, J.A., Gundlach, IV, W., Hampel, K.J., Glick, G.D., Walter, N.G., Major, F., Burke, J.M. The hairpin ribozyme substrate binding domain: A highly constrained D-shaped conformation. J. Mol. Biol. 2001, 307:51-65.
Pinard, R., Lambert, D., Walter, N.G., Heckman, J.E., Major, F., and Burke, J.M. Structural basis for the guanosine requirement of the hairpin ribozyme. Biochemistry 1999, 38:16035-16039.