Sequence-dependent folding and unfolding of ligand-bound purine riboswitches

Riboswitch regulation of gene expression requires ligand-mediated RNA folding. From the fluorescence lifetime distribution of bound 2-aminopurine ligand, we resolve three RNA conformers (C(o), C(i), C(c)) of the liganded G- and A-sensing riboswitches from Bacillus subtilis. The ligand binding affinities, and sensitivity to Mg(2+), together with results from mutagenesis, suggest that C(o) and C(i) are partially unfolded species compromised in key loop-loop interactions present in the fully folded C(c). These data verify that the ligand-bound riboswitches may dynamically fold and unfold in solution, and reveal differences in the distribution of folded states between two structurally homologous purine riboswitches: Ligand-mediated folding of the G-sensing riboswitch is more effective, less dependent on Mg(2+), and less debilitated by mutation, than the A-sensing riboswitch, which remains more unfolded in its liganded state. We propose that these sequence-dependent RNA dynamics, which adjust the balance of ligand-mediated folding and unfolding, enable different degrees of kinetic discrimination in ligand binding, and fine-tuning of gene regulatory mechanisms.