Atomic-scale characterization of conformational changes in the preQ1 riboswitch aptamer upon ligand binding

Riboswitches are mRNA structural elements that act as intracellular sensors of small-molecule metabolites. By undergoing conformational changes capable of modulating translation or terminating transcription, riboswitches are able to play a role in regulating the concentration of essential metabolites in the cell. Computer-guided fluorescence experiments were carried out to interrogate molecular dynamics and conformational changes in the minimal riboswitch aptamer that binds 7-aminomethyl-7-deazaguanine (preQ1). Our combined experimental results and computational analysis suggest that the preQ1 riboswitch apo form is structured but shows no evidence of a ligand-binding pocket. Simulations of the apo and bound forms indicate a large conformational change is triggered by the breaking of the Watson–Crick base pairing of nucleotides G11 and C31 upon preQ1 removal, followed by collapse of the pocket due to interfering π-stacking. Computational predictions of local aptamer dynamics were validated by fluorescence experiments employing 2-aminopurine substitutions. In-line probing reactions confirmed that fluorophore-labeled riboswitches retain similar higher-order structural features as the unlabeled aptamer upon ligand binding, although their affinity for the ligand is reduced by the introduction of the fluorescent reporter.

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► Bacillus subtilis preQ1 apo form of the riboswitch aptamer is structured.
► S2 loop undergoes a dramatic conformational change as G11–C31 come together into a Watson–Crick base pair.
► 570 ns (apo) and 470 ns (bound) preQ1 riboswitch molecular dynamics trajectories.
► Computational predictions of aptamer dynamics validated by fluorescence spectroscopy using 2-aminopurine substitutions.