Single-molecule studies of the lysine riboswitch reveal effector-dependent conformational dynamics of the aptamer domain.
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 Bacillus subtilis lysC aptamer domain. These techniques permit measurement of the dissociation constant (K(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 (K(D,apparent)) of 0.25(9) mM at near physiological ionic strength, which differs markedly from previous reports.
Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 80309, United States.
Larry R
LR
Fiegland
Andrew D
AD
Garst
Robert T
RT
Batey
David J
DJ
Nesbitt
D052157:Aptamers, Nucleotide / Q000737:chemistry* / Q000378:metabolism; D001412:Bacillus subtilis / Q000235:genetics; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D007700:Kinetics; D008024:Ligands; D008239:Lysine / Q000235:genetics* / Q000378:metabolism; D009690:Nucleic Acid Conformation / Q000187:drug effects*; D012329:RNA, Bacterial / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D058928:Riboswitch
D052157:Aptamers, Nucleotide; D008024:Ligands; D012329:RNA, Bacterial; D012333:RNA, Messenger; D058928:Riboswitch; D008239:Lysine