Single-Molecule Studies of the Lysine Riboswitch Reveal Effector-Dependent Conformational Dynamics of the Aptamer Domain

Abstract

The lysine riboswitch is a cis-acting RNA genetic regulatory element found in the leader sequence of bacterial mRNAs coding for proteins related to biosynthesis or transport of lysine. Structural analysis of the lysine-binding aptamer domain of this RNA has revealed that it completely encapsulates the ligand and therefore must undergo a structural opening/closing upon interaction with lysine. In this work, single-molecule fluorescence resonance energy transfer (FRET) methods are used to monitor these ligand-induced structural transitions that are central to lysine riboswitch function. Specifically, a model FRET system has been developed for characterizing the lysine dissociation constant as well as the opening/closing rate constants for the <i>Bacillus subtilis lysC</i> aptamer domain. These techniques permit measurement of the dissociation constant (<i>K</i>_D) for lysine binding of 1.7(5) mM and opening/closing rate constants of 1.4(3) s^–1 and 0.203(7) s^–1, respectively. These rates predict an apparent dissociation constant for lysine binding (<i>K</i>_D,apparent) of 0.25(9) mM at near physiological ionic strength, which differs markedly from previous reports