Structural transitions and thermodynamics of a glycine-dependent riboswitch from Vibrio cholerae.
Riboswitches are complex folded RNA domains found in noncoding regions of mRNA that regulate gene expression upon small molecule binding. Recently, Breaker and coworkers reported a tandem aptamer riboswitch (VCI-II) that binds glycine cooperatively. Here, we use hydroxyl radical footprinting and small-angle X-ray scattering (SAXS) to study the conformations of this tandem aptamer as a function of Mg(2+) and glycine concentration. We fit a simple three-state thermodynamic model that describes the energetic coupling between magnesium-induced folding and glycine binding. Furthermore, we characterize the structural conformations of each of the three states: In low salt with no magnesium present, the VCI-II construct has an extended overall conformation, presumably representing unfolded structures. Addition of millimolar concentrations of Mg(2+) in the absence of glycine leads to a significant compaction and partial folding as judged by hydroxyl radical protections. In the presence of millimolar Mg(2+) concentrations, the tandem aptamer binds glycine cooperatively. The glycine binding transition involves a further compaction, additional tertiary packing interactions and further uptake of magnesium ions relative to the state in high Mg(2+) but no glycine. Employing density reconstruction algorithms, we obtain low resolution 3-D structures for all three states from the SAXS measurements. These data provide a first glimpse into the structural conformations of the VCI-II aptamer, establish rigorous constraints for further modeling, and provide a framework for future mechanistic studies.
Lipfert
Jan
J
Department of Physics, Stanford University, Stanford, CA 94305, USA.
Das
Rhiju
R
Chu
Vincent B
VB
Kudaravalli
Madhuri
M
Boyd
Nathan
N
Herschlag
Daniel
D
Doniach
Sebastian
S
D052157:Aptamers, Nucleotide / Q000737:chemistry* / Q000235:genetics; D001483:Base Sequence; D005998:Glycine / Q000378:metabolism*; D017665:Hydroxyl Radical / Q000378:metabolism; D008274:Magnesium / Q000494:pharmacology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation / Q000187:drug effects; D009691:Nucleic Acid Denaturation / Q000187:drug effects; D012329:RNA, Bacterial / Q000737:chemistry* / Q000235:genetics; D053838:Scattering, Small Angle; D012997:Solvents; D013816:Thermodynamics; D014734:Vibrio cholerae / Q000737:chemistry*; D014961:X-Ray Diffraction
D052157:Aptamers, Nucleotide; D012329:RNA, Bacterial; D012997:Solvents; D017665:Hydroxyl Radical; D008274:Magnesium; D005998:Glycine