Folding and dynamics of a preQ1-binding riboswitch

Riboswitches are gene regulatory elements that predominantly occur in the untranslated leader regions of bacterial mRNA and that recognize specific metabolites. In recent years, much progress has been achieved in characterizing metabolite-bound riboswitch states with high-resolution structures. However, the research field would benefit from an increased number of dynamics analyses, to help correlate the RNA/ligand-binding events with their downstream effects on gene regulation in cells. In that light, this research program aims at a better understanding of the folding and dynamics of the preQ1 class II (preQ1 cII) riboswitch. This riboswitch specifically binds 7-aminomethyl-7-deazaguanine (pre-queuosine1 ; preQ1 ), a precursor of the modified nucleoside queuosine that occurs in tRNA, through a pseudoknot fold. The folding of the riboswitch will be characterized using 2-aminopurine fluorescence and selective 2-hydroxyl acylation analyzed by primer extension (SHAPE) probing experiments, while the dynamics will be addressed by single-molecule fluorescence resonance energy transfer (smFRET) experiments. Furthermore, isothermal titration calorimetry (ITC) will also be used to support the aim. The combination of experiments proposed in this application will likely allow us to gather enough information on preQ1 cII folding and dynamics to present a viable mechanism for preQ1 ligand-binding and the concomitant ligand-induced folding pathway responsible for gene regulation by this riboswitch. The unique information thereby gathered at the molecular level is beneficial to the general RNA community, allowing a greater understanding of the dynamics involved in RNA folding. Additionally, the research plan highlighted in this proposal will contribute to ongoing efforts to employ riboswitches as antibiotic targets, as well as to engineer artificial cell regulation systems.