Project Summary The central theme of this proposal is structural and mechanistic studies of nucleic acids, proteins, and their interactions. The two broad topics that will serve as foci for our future research are: 1) the structure and mechanism of topoisomerases, and 2) the structure and mechanism of non-coding RNA molecules, particularly T-box riboswitches. The aim of our work is to discover and explore paradigms that help to illuminate the structure and mechanism of biological molecules. Topoisomerases are ubiquitous proteins found across all three domains of life. They are involved in several cellular processes and the importance of their cellular role is underscored by the fact that they are the target of several cancer chemotherapeutic agents and antibiotics. The study of the structure, mechanism, and function of topoisomerases not only furthers our understanding at many different levels of proteins that interact with DNA and alter its topological properties, but also provides important information to aid in the design of new therapeutic agents. We will continue our comprehensive studies of topoisomerases with an emphasis on three large and complementary sub-projects: i) Single molecule studies of type IA topoisomerases, ii) Structural studies of topoisomerases on natural substrates, and iii) Structural studies of gyrase in complex with mini- circle DNA. These sub-projects will help answer crucial questions that are needed to advance the field to the next level. Riboswitches are ligand-specific cis-regulatory RNA elements found in untranslated regions of mRNAs that respond to defined external signals to affect transcription or translation of downstream genes. Bacterial T- box riboswitches represent a unique class of riboswitches that do not bind small molecule ligands, instead they recognize and bind tRNA molecules and sense directly their aminoacylation state. They represent an excellent paradigm to understand RNA structure and architecture, recognition of RNA molecules by other RNA molecules, RNA-based gene regulation, and the evolution of gene regulatory function from the ancient RNA world. We will continue and expand our studies of T-box riboswitches by concentrating in three subprojects: i) Single molecule studies of translational T-box riboswitches, ii) Structural and single molecule studies of typical T-box riboswitches, and iii) Structural studies of tandem T-box riboswitches. These three sub-projects will advance our understanding of T-box riboswitches and non-coding RNA molecules, molecules that are still not fully understood at the atomic level.