In the past year, we have made important progress in understanding the molecular basis of action of the cyclic diguanylate (c-di-GMP) riboswitch. All known bacteria, including pathogens such as Staphylococcus aureus and Vibrio cholerae use the second messenger c-di-GMP to transition from the motile to the biofilm lifestyles. The c-di-GMP riboswitch is the master sensor of this second messenger, controlling the many pathways that need to be coordinately regulated for biofilm formation. We have previously determined the crystal structure of the c-di-GMP riboswitch from V. cholerae bound to the second messenger. Now, we have elucidated the dynamic process of c-di-GMP binding through single-molecule FRET techniques, and find that RNA tertiary interactions distal to the c-di-GMP preorganize the riboswitch so that it can respond to rapid changes in the intracellular concentration of the second messenger. This work lays the foundation for the discovery of small molecules that can adversely affect the preorganization of the RNA, and thus serve to disable biofilm formation by bacteria at the level of gene expression. We have also discovered and characterized bacterial RNA binding proteins that associate with an RNA structural motif called the K-turn. The K-turn is essential for stability and function of several bacterial riboswitches. Through crystallographic and biophysical means, we have shown that the protein YbxF, of previously unknown function, specifically binds to K-turns, and modulates the affinity of several riboswitches for their cognate ligands. Because YbxF is only present in bacteria, not eukaryotes, the molecular interface of YbxF and K-turns can be a target for small molecule drugs that would impair riboswitch function without affecting the human host.
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