John Burke, Ph.D.
Professor of Microbiology and Molecular Genetics
Research Program: Genome Stability & Expression
VCC Membership Level: Member Scientist
Contact Information
220B Stafford Hall
95 Carrigan Avenue
University of Vermont
Burlington, VT 05405
ph: (802) 656-8503
f: (802) 656-8749
John.Burke@uvm.edu
Biography
Dr. Burke received his Ph.D. in 1983 from the Biology Department of the Massachusetts Institute of Technology, where he studied intron organization in Neurospora mitochondrial genes with Tom RajBhandary. He was assistant professor of chemistry at Williams College before joining the UVM College of Medicine faculty in 1988.
Research
The research program in Dr. Burke's laboratory focuses on catalytic RNA molecules, or ribozymes, employing both hairpin and hammerhead ribozymes. His projects focus on four major aspects of ribozyme research: (1) molecular structures, (2) RNA conformational changes, (3) catalytic mechanisms, and (4) applying ribozyme technology to gene therapy and functional genomics. He employs a wide range of complementary experimental methods, including combinatorial selection, fluorescence spectroscopy, computational biology, kinetics, and experimental virology. Together, these experimental approaches provide Dr. Burke's laboratory with the ability to conduct a comprehensive analysis of ribozyme structure, activity, and applications.
The hammerhead and hairpin ribozymes both catalyze RNA cleavage reactions through a phosphoester transfer pathway that does not involve metal ions in the reaction mechanism. Until very recently, biochemical and biophysical studies had used the hairpin ribozyme system. Dr. Burke showed that the hairpin ribozyme undergoes a dramatic conformational change during formation of an active complex in which the active site is buried in a solvent-inaccessible zone within the three-dimensional structure. UV-crosslinking, combinatorial selection, and hydroxyl radical footprinting experiments provided data that Dr. Burke then used in computational studies to develop and test models of the active site, and to demonstrate that G8 is a key player in reaction chemistry. Nearly all of the important features of Dr. Burke's models were confirmed by recent crystallographic studies of the hairpin ribozyme-substrate complex.
Very recently, Dr. Burke's laboratory uncovered evidence that the hammerhead and hairpin ribozymes may be much more similar than was previously suspected. UV crosslinking, base substitutions, and hydroxyl radical footprinting have allowed us to identify and analyze a hammerhead structure that appears to be reactive, and is quite different from the hammerhead structures that have been solved by crystallography. The working hypothesis is that the crystal structures represent a ground state, and that a significant conformational change is required to generate an active complex. His laboratory has begun an intensive analysis of the hammerhead, and believe that there may be striking similarities between the hammerhead and hairpin ribozyme active sites and catalytic mechanisms.
Dr. Burke has also shown that engineered ribozymes can be effectively used to inhibit viral replication in mammalian cells. This has been a very daunting task, since it has required his lab to learn how to identify optimal target sites within viral RNA molecules, stably express small ribozymes, and localize them to specific subcellular sites. Recently, they have succeeded in using hairpin ribozymes to selectively inhibit the replication of Sindbis virus, a member of the alphavirus family which replicates very aggressively within the cytoplasm. Current work focuses on (i) optimizing ribozyme inhibition, (ii) using genetics to prove the site and mechanism of antiviral activity, and (iii) extending this technology to develop therapeutic strategies for significant infectious and genetic diseases, and to the area of functional genomics.
Recent Publications
Gaur S, Heckman JE, Burke JM. 2008. Mutational inhibition of ligation in the hairpin ribozyme: Substitutions of conserved nucleobases A9 and A10 destabilize tertiary structure and selectively promote cleavage. RNA, 14, 55-65. PMCID: PMC2151026.
Lambert D, Burke JM. 2007. Finding the Hammerhead Ribozyme Active Site. In Ribozymes and RNA Catalysis, Lilley, D.M.J. and Eckstein, F. Eds., 37-47.
Lambert D, Heckman JE, Burke JM. 2006. Three conserved guanosines approach the reaction site in native and minimal hammerhead ribozymes. Biochemistry, 45, 7140-7147.
Lambert D, Heckman JE, Burke JM. 2006. Cation-specific structural accommodation within a catalytic RNA. Biochemistry, 45, 829-838.
Han J, Burke JM. 2005. Model for general acid-base catalysis by the hammerhead ribozyme: pH-activity relationships of G8 and G12 variants at the putative active site. Biochemistry, 44, 7864-7870.
Heckman JE, Lambert D, Burke JM. 2005. Photocrosslinking detects a compact active structure of the hammerhead ribozyme. Biochemistry, 44, 4148-4156.
Zhang Z, Burke JM. 2005. Inhibition of viral replication by ribozyme: Mutational analysis of the site and mechanism of antiviral activity. J. Virol., 79, 3728-3736. PMCID: PMC1075678.
Pinard R, Lambert D, Pothiawala G, Major F, Burke JM. 2004. Modifications and deletions of helices within the hairpin ribozyme-substrate complex: an active ribozyme lacking helix 1. RNA 10, 395-402. PMCID: PMC1370935.
Other Key Publications
Sargueil B, Hampel KJ, Lambert D, Burke JM. 2003. In vitro selection of second-site revertants analysis of the hairpin ribozyme active site. J. Biol. Chem. 278, 52783-52791.
Hampel KJ, Burke JM. 2003. Solvent protection of the hammerhead ribozyme in the ground state: Evidence for a cation-assisted conformational change leading to catalysis. Biochemistry 42, 4421-4429.
Burke JM. 2002. Hairpin and hammerhead ribozymes: How different are they? Biochem. Soc. Trans. 30, 1116-1119.
Seyhan A, Vitiello D, Shields MT, Burke JM. 2002. Ribozyme inhibition of alphavirus replication. J. Biol. Chem. 277, 25957-25962.
Pinard R, Hampel KJ, Heckman JE, Lambert D, Chan PA, Major F, Burke JM. 2001. Functional involvement of G8 in the hairpin ribozyme cleavage mechanism. EMBO J. 20, 6434-6442. PMCID: PMC125305.
