The rise of antibiotic resistance calls for immediate focus on identifying novel drug targets. Riboswitches are a distinct class of RNA that have already been validated as drug targets. They take part in gene regulation in direct response to specific small-molecule effectors' levels. Studies on riboswitches have mostly focused on their ligand binding abilities in vitro, outpacing our understanding of the underlying mechanisms that link effector-binding to gene-regulation. To convincingly relate RNA structure to function, I analyzed the preQ1-II riboswitch: a well-characterized riboswitch that recognizes the metabolite pre-queuosine1 (preQ1). I used an RNA chemical-modification analysis called in cell (ic)SHAPE to compare flexibility changes of individual nucleotides in response to preQ1. The gene-OFF conformational state data showed excellent correlation with our bound-state crystal structure. We then developed a reporter assay in which GFPuv is controlled by a preQ1-II riboswitch to study its gene regulatory function. Added effector showed a 10-fold repression of GFPuv expression, consistent with binding studies done by Isothermal Titration Calorimetry (ITC). The functional relevance of in vitro observations thus established, I sought to identify molecular interactions that connect preQ1 binding to gene-regulation. We used our reporter assay and ITC along with site-directed mutagenesis to study specific nucleobase interactions hypothesized to be on molecular signal-transduction pathways. Analyses were conducted on 15 mutants flanking the preQ1-binding pocket. Several conserved elements of the riboswitch, like the ?A-minor' bases and the helix P4 were found dispensable for effector-sensing but are playing an immense role in gene-regulation. I also observed several interactions that are essential for gene-regulation inside live cells but were not for ligand-binding. The results helped shed light on how riboswitches function in their natural environment inside cells. I also discuss the nucleoside-dynamics and structural changes occurring to the preQ1-I riboswitch upon ligand-binding. I used chemical probing methods like DMS and SHAPE and found bases proximal to the ligand-binding site undergoing a change in their conformation or flexibility upon effector-binding. Together with crystal structure, computational studies and 2AP-fluorescence studies, we confirmed that the dynamic L2 loop in the apo-state riboswitch is key to functioning of the RBS for effective gene-regulation.