Single-Molecule Studies
of the Lysine Riboswitch Reveal
Effector-Dependent Conformational Dynamics of the Aptamer Domain
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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><sub>D</sub>) for lysine binding of 1.7(5) mM and opening/closing
rate constants of 1.4(3) s<sup>β1</sup> and 0.203(7) s<sup>β1</sup>, respectively. These rates predict an apparent dissociation
constant for lysine binding (<i>K</i><sub>D,apparent</sub>) of 0.25(9) mM at near physiological ionic strength, which differs
markedly from previous reports