Project Summary. A widespread mechanism of gene regulation in bacteria is by a group of noncoding RNA elements called a riboswitch. These are cis-acting elements found in the leader sequence of mRNAs and regulate gene expression through their ability to directly bind a specific effector molecule to a highly-structured aptamer domain. Effector binding to the aptamer domain is communicated to a downstream secondary structural switch in the expression platform that instructs the expression machinery (generally RNA polymerase or the ribosome). In a broad spectrum of bacteria, particularly Firmicutes and Fusobacteria, central metabolic pathways including purine, amino acid, and cofactor biosynthesis and transport are regulated by riboswitches. Furthermore, genes essential for survival or virulence are under riboswitch control in numerous medically important pathogens including Listeria monocytogenes, Staphylococcus aureus, Pseudomonas aeruginosa, Clostridium difficile, and Mycobacterium tuberculosis making them of great interest as novel targets for antimicrobial therapeutics. Of equal importance, riboswitches are powerful model systems for understanding various aspects of RNA biology including structure, folding and mechanisms of regulatory activity along with developing tools and methodologies for designing small molecules that target other RNAs of medical interest. Towards the long-term goal of developing a molecular understanding of how RNA interacts with small molecules and the mechanisms it uses to regulate gene expression, we are using purine- and cobalamin- binding riboswitches as model systems. This proposal details a set of interconnected specific aims that addresses fundamental questions related to these research goals: (1) mapping sequence and structural features of the expression platform beyond the structural switch crucial for efficient ligand-dependent regulatory activity, (2) understanding how structural ?modules? that mediate higher-order tertiary structure contribute to rapid and efficient folding of the aptamer domain, and (3) investigating the plasticity of aptamer domains through their ability to recognize different ligands. To address these questions, a combination of structural (x-ray crystallography) biophysical (calorimetry and stopped-flow kinetics), biochemical (chemical footprinting), genetic and molecular biological (in vivo and in vitro activity assays) and bioinformatics/computational approaches will be combined to study the structure-regulatory activity linkage in riboswitches. Significantly, this proposal adopts a ?function-first? research strategy, as opposed to the ?structure-first? approach that dominates current research into riboswitches to make a stronger connection between RNA structure and function. A deeper knowledge of how RNA specifically interacts with small molecules that affects its structure and activity will contribute to ongoing efforts to develop a new generation of therapeutics that target non-protein coding RNAs in bacteria and eukaryotes.