Molecular Basis of RNA-Mediated Gene Regulation on the Adenine Riboswitch by Single-Molecule Approaches

The adenine-specific pbuE riboswitch undergoes metal ion-dependent folding that involves a long-range tertiary loop–loop interaction between two stem loops. Fluorescence resonance energy transfer (FRET) and single-molecule FRET studies demonstrate the ability of the loops to interact in the absence of the ligand. Although the riboswitch can fold in the absence of adenine, ligand binding stabilizes this folded conformation by increasing the folding and decreasing the unfolding rates of the riboswitch. The presence of the ligand also decreases the magnesium ion concentration required to promote the loop–loop interaction. Single-molecule FRET studies demonstrate that individual aptamer molecules exhibit great heterogeneity in the rates of folding and unfolding, which is reduced in the presence of adenine. Moreover, single-molecule FRET proposes that riboswitch folding proceeds through a complex landscape that involves a discrete intermediate.