Riboswitches are folded RNA domains that bind specific metabolites and act as regulators of gene expression. The bound metabolites are the biosynthetic products or substrates of the downstream genes, resulting in an economical feedback loop mechanism for altering gene expression in response to physiological needs. Riboswitches are composed of two functional elements: a metabolite binding domain and a downstream expression platform. Conformational changes in the riboswitch induced by metabolite binding result in modulation of gene expression by altering transcriptional elongation or translational initiation. While riboswitches are prevalent, the structural basis of metabolite binding and the nature of the effector promoted conformational changes are largely unknown.
This project focuses on two complex riboswitches: the glmS and the glycine riboswitch. Both of these RNAs demonstrate functions that go beyond simple metabolite binding. The glycine riboswitch is an upregulator of gene expression and is found as a tandem repeat of two closely related sequences, each of which is able to independently bind glycine. In tandem the aptamers bind cooperatively. The result is a "digital" riboswitch that is exquisitely sensitive to the glycine concentration. This riboswitch demonstrates that RNA, like protein, can achieve cooperative allosteric binding of a small molecule. How it does this is unknown. The glmS riboswitch functions as a metabolite dependent ribozyme. The glmS riboswitch is responsive to the metabolite glucosamine-6-phosphate (Gln6P) resulting in down regulation of glmS gene expression. Gln6P binding induces self-cleavage of the RNA at a specific residue 5' of the riboswitch sequence. It is unclear how the RNA and Gln6P interact to achieve such activity.
The overall goal of this project is to understand the structural and chemical basis of these complex riboregulators. The research will address the following questions: How do these ranks bind their small molecule effectors? How does the glmS RNA fold to create an active site for catalysis? Does the Gln6P simply induce a conformational change to activate the RNA, or does one of its functional groups participate directly in the chemical reaction? How does the glycine riboswitch achieve cooperative binding of two glycine molecules? Do the two domains physically interact with each other, or is the cooperatively less direct? To answer these questions, the project will use a combination of organic synthesis, RNA biochemistry and X-ray crystallography methods.
Broader Impacts: This project will continue to provide a nurturing environment for research training of students, particularly of individuals from minority groups traditionally underrepresented in the sciences. The Principle Investigator serves as the Director of Undergraduate Studies in the Molecular Biophysics and Biochemistry Department at Yale University, where he mentors 35-50 science majors per year. He will teach courses in Biochemistry, Mechanistic Enzymology and Scientific Logic at the undergraduate and graduate levels. The PI will continue to serve as the faculty advisor to the student campus chapter of the ASBMB, which last year initiated the first annual Yale Undergraduate Science Forum that included oral and poster presentations of the student's research accomplishments during the academic year.
Organic and Macromolecular Chemistry Program, Genes and Genome Systems Cluster, and Biomolecular Systems Cluster support this project jointly.
