Dr. Joyce E. Heckman

Assistant Professor

Catalytic RNA

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
The second major effort in our group involves the expression of hairpin ribozymes in cells. These ribozymes can be engineered to target specific sequences in the genome of an RNA virus, and by cleaving the RNA in an infected cell, inhibit the replication of the virus. Using Sindbis virus (a positive- strand alphavirus with a very rapid replication cycle) as a model system, we are exploring means of expressing large numbers of ribozymes in cells, localizing them to the cytoplasm where the virus replicates, and optimizing their activity in order to disrupt the reproduction of this aggressive virus. We also are undertaking a project to prove whether site-specific RNA cleavage by ribozyme is actually the mechanism of viral inhibition. This effort involves mutating the viral RNA sequence at the cleavage site to prevent ribozyme function, then generating compensating mutations in the ribozyme to see whether they restore cleavage and the inhibition of viral infection.

BACKGROUND

Dr. Heckman received her Ph.D. from The Massachusetts Institute of Technology in 1976 under the direction of Professor U.L. RajBhandary, and also did postdoctoral work in the same group at MIT. She was a faculty member in the Chemistry Department at Indiana University 1981-1988. She joined the UVM faculty in 1994.

SELECTED PUBLICATIONS

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.

Pinard, R., Heckman, J.E. and Burke, J.M. Alignment of the two domains of the hairpin ribozyme-substrate complex defined by inter-domain photoaffinity crosslinking.

Yu, Q., Pecchia, D.B., Kingsley, S.L., Heckman, J.E. and Burke, J.M. Cleavage of highly structured viral RNA molecules by combinatorial libraries of hairpin ribozymes.