Observation of preQ₁-II riboswitch dynamics using single-molecule FRET

PreQ₁ riboswitches regulate the synthesis of the hypermodified tRNA base queuosine by sensing the pyrrolopyrimidine metabolite preQ₁. Here, we use single-molecule FRET to interrogate the structural dynamics of apo and preQ₁-bound states of the preQ₁-II riboswitch from Lactobacillus rhamnosus. We find that the apo-form of the riboswitch spontaneously samples multiple conformations. Magnesium ions and preQ₁ stabilize conformations that sequester the ribosome-binding site of the mRNA within the pseudoknotted structure, thus inhibiting translation initiation. Our results reveal that folding of the preQ₁-II riboswitch is complex and provide evidence favoring a conformational selection model of effector binding by riboswitches of this class.