Ligand-directed dynamics of adenine riboswitch conformers.
Riboswitches harness the structural and dynamic sophistication of RNA to coordinate specific ligand recognition with changes in gene expression. Design of molecules to manipulate riboswitch responses relies on our understanding of their RNA−ligand interactions. Here we demonstrate that for the adenine (A) riboswitch (ARNA) these interactions are highly dynamic. Given that 2-aminopurine (Ap) mimics A in its interactions with ARNA, we use the fluorescence lifetime of Ap to interrogate individual Ap-ARNA conformers (dynamic exchange times > ∼10 ns). Counter to predictions of two state and induced fit models, the ligand-bound A riboswitch is not a single, highly ordered structure: We detect at least three distinct Ap-ARNA conformers in ensemble solution. Their distribution indicates that they are not high-energy RNA folding intermediates but are instead energetically similar (ΔG < 1 kcal mol-1) conformers whose thermal stability, ligand, and Mg2+ binding affinity differ substantially. Our experimental charac...