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, which binds its ligand cooperatively; 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.
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. We are applying biophysical techniques with the help of synthetic chemistry to determine how the ribosome catalyzes this fundamental reaction of biology. We have determined the transition state for peptide bond formation using a combination of kinetic isotope effects, linear-free energy relationships, and transition state analogs. We are extending our studies to peptide release, the final reaction of protein synthesis. Again aided by synthetic chemistry, we are using kinetic isotope effects and transition state analogs to determine the structure of the transition state for this reaction and the relative positioning of ribosomal functional groups. We are also studying a third chemical reaction on the ribosome - the cleavage of mRNA by the ribosome-dependent endonuclease RelE. 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 biochemical and genetic approaches to establish how RelE residues and the ribosome each contribute to this cleavage reaction.
Endophytic Fungi. Endophytic fungi are a diverse niche of organisms that produce a wide variety of useful molecules. We isolate and identify new endophytes by traveling to the world’s rainforests and bringing samples back to Yale to culture. A typical collecting expedition yields hundreds of isolates in culture, roughly 20% of which are new species. We then screen these new organisms to search for novel activities. In particular, we focus on phenotypes that would be useful for the production of biofuels. These include being able to degrade alternative polymers that could potentially be biofuel feedstocks, being resistant to the toxicity associated with exposure to biofuels, and the production of novel volatile molecules with properties that would be useful as biofuels. We study these processes using a wide variety of techniques, often including metabolic labeling, gas chromatography mass spectrometry and high-throughput sequencing, seeking to understand these novel phenotypes at chemical and genetic level.