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Riboswitches. Riboswitches are noncoding RNAs that regulate the expression of genes in response to the presence of small molecules. We use biophysical and biochemical techniques aided by synthetic organic chemistry to study the structures and functions of these important RNAs. We have biochemically characterized and solved the X-ray structures of several riboswitch classes: the glycine riboswitch; two classes of c-di-GMP riboswitches, which bind an important second messenger and function in cell signaling; and the glmS ribozyme, which controls gene expression by self-cleaving in the presence of its ligand. We are currently pursuing several approaches in order to more fully understand these macromolecules. Efforts include determining the atomic resolution structures of several new riboswitch classes as well as alternative complexes, investigating intramolecular interactions in the glycine riboswitch to understand the molecular basis of cooperativity in an RNA system, designing and testing ligand analogs to probe small molecule-RNA interactions for the design of potential drug candidates and performing in vivo studies of riboswitch variants to determine their regulatory mechanisms.
Fluoride Homeostasis and Toxicity. Riboswitches can also provide valuable insights into biology, as they imply a connection between a small molecule ligand and a downstream gene. In one dramatic example, the fluoride riboswitch indicated a role for a membrane protein of unknown function in fluoride biology. Now, multiple fluoride channels have been discovered that are responsible for maintaining fluoride below toxic levels in both prokaryotic and eukaryotic cells. We are investigating the mechanism, localization, and regulation of fluoride transport by FEX, the yeast fluoride channel. We have shown that FEX is constitutively expressed and is necessary to prevent toxicity even at the fluoride levels found in tap water. We are also searching for other eukaryotic homologs of FEX through a combination of bioinformatics and molecular biology.
Protein Synthesis. Protein synthesis in all organisms is catalyzed by the ribosome, a large complex of RNA and protein. Crystallographic studies have revealed that the peptidyl transferase center, where the chemical reactions of protein synthesis take place, is composed exclusively of RNA. Therefore, this ancient and essential process is catalyzed not by protein but by RNA: the ribosome is a ribozyme. Using a combination of kinetic isotope effects, linear free-energy relationships, and transition state analogs, we showed that the ribosome alters the reaction pathway to catalyze peptide bond formation at a rate that can sustain life. We are now extending our studies to the myriad of other processes that occur on the ribosome. In particular, we are investigating the mechanism of RelE, an mRNA endonuclease. RelE is structurally similar to other endoribonucleases, but it lacks conserved catalytic residues and it is only active in the context of the ribosomal A-site. We are using biochemistry and genetics, aided by chemical synthesis, to establish how RelE residues and the ribosome each contribute to this cleavage reaction.