John M. Burke, PhD
Professor

Research Interests

The research program in my lab focuses on catalytic RNA molecules, or ribozymes, employing both hairpin and hammerhead ribozymes. Our projects focus on four major aspects of ribozyme research: (1) molecular structures, (2) RNA conformational changes and (3) catalytic mechanisms. We employ a wide range of complementary experimental methods, including combinatorial selection, fluorescence spectroscopy, computational biology, kinetics, and experimental virology. Together, these experimental approaches provide us 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, all of our biochemical and biophysical studies had used the hairpin ribozyme system. We 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 we 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 our models were confirmed by recent crystallographic studies of the hairpin ribozyme-substrate complex.

Very recently, we have 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. Our working hypothesis is that the crystal structures represent a ground state, and that a significant conformational change is required to generate an active complex. We have 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.

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