Our group is broadly interested in the chemistry and biochemistry of nucleic acids with particular emphasis on RNA and RNA catalysis. The laboratory integrates areas of organic chemistry, physical chemistry, enzymology and molecular biology to gain a fundamental understanding of nucleic acid structure and mechanisms of RNA catalysis. Using the principles and techniques of organic chemistry and molecular biology, we manipulate the structure of RNA molecules at precise locations in ways that are designed to answer very specific questions about biological function.
 
We employ these approaches toward gaining a fundamental understanding of the role that divalent metal ions play in phosphoryl transfer reactions that occur during RNA splicing, an important step in genetic expression. One experimental system that we are using to address these issues is the self-splicing intervening sequence RNA of the ciliated protozoan Tetrahymena. Shortened forms of this RNA can act as enzymes, catalyzing the sequence specific cleavage of RNA and DNA substrates with multiple turnover. We have used sulfur substitution of the oxygen substituents on the phosphoryl group undergoing transfer to reveal the transition state interactions between the ribozyme and the scissile phosphate.

Another area of interest is the development of new methods and model systems for studying RNA molecules. For example, we have recently designed a series of nucleoside analogues in which the 2'-beta-hydrogen atom is replaced by CH3, CH2F, CHF 2, or CF 3 . These analogues provide a systematic way to perturb the acidity of the 2'-OH group, thereby allowing us to probe the all important role of this functional group in RNA mediated biological processes.

We also investigate the mechanism of protein-RNA interactions using small ribonuclease restrictocin as a probe. Restrictocin is a small protein (149 amino acids) that is able to cross the cell membrane and cleave the 23–28S ribosomal RNA at a single phosphodiester bond. The cleavage site resides in a region of the ribosomal RNA known as the sarcin/ricin loop (SRL), which folds into a tetraloop motif and a bulged-G motif and participates in the binding of elongation factors during protein synthesis by the ribosome. Considering that the 28S ribosomal RNA contains thousands of phosphodiester bonds, the apparent specificity of this ribonuclease is remarkable. This single cleavage event inactivates the ribosome and consequently abolishes its ability to carry out protein synthesis, which ultimately leads to death of the cell. This system has a broad significance in biology as a model system to study RNA-protein interactions, which are ubiquitous and mediate numerous important events during gene expression. Remarkably, few functional studies have been reported on this protein. Our initial focus will be to determine the dynamic changes that occur in the SRL when it binds to restrictocin and to elucidate the energetic contributions that enzyme-RNA substrate contacts play in cleavage-site recognition and catalysis.

Affiliations:

The University of Chicago, Department of Chemistry

The University of Chicago, Department of Biochemistry and Molecular Biology

HHMI