Fig. 4: Kinetics of ligand binding to SAM-VI RNA.
From: SAM-VI riboswitch structure and signature for ligand discrimination
a Sequence and secondary structure of the 2-aminopurine (Ap) modified RNA used for fluorescence spectroscopic experiments. b The nucleoside U39 (in red) that was selected for Ap replacement is located close to the binding pocket and directed outwards. c Real time fluorescence traces for SAM-VI riboswitch complex formation, upon Mg2+ and ligand additions. d Fluorescence changes upon titration of Ap labeled SAM-VI riboswitch variant (wt U39Ap) with ligand SAM; fluorescence emission spectra (λex = 308 nm) from 330 to 450 nm of the wt U39Ap variant for each ligand concentration. e Normalized fluorescence intensity of the wt U39Ap variant plotted as a function of SAM ligand concentration. The graph shows the best fit to a singleÂsite binding model (see Methods). Changes in fluorescence (F-F0) were normalized to the maximum fluorescence measured in saturating concentrations of the SAM ligand. The obtained Kd (Ap) values (50 mM KMOPS, pH 7.5, 100 mM KCl, 2 mM MgCl2, 293 K) were slightly higher compared to the Kd values obtained from ITC titration experiments that were performed at 10 mM Mg2+ concentrations. f, g Same as d, e but for titration of the U6C U39Ap variant with SAM ligand. For further details, see Methods. h Stopped-flow fluorescence spectroscopy was used to monitor the kinetics of SAM-VI riboswitch complex formation using wild-type (wt) and U6C SAM-VI RNAs. Exemplary fluorescence traces are depicted; conditions: 0.3 μM RNA, 100 mM KCl, 50 mM MOPS, pH 7.5, 293 K. Ligands: 2 mM MgCl2, 1.8 μM ligand. i The mean kobs values determined from three independent experiments with the corresponding error bars are plotted against the concentration of SAM and subjected to a linear fit. The slope of the plot yields the rate constant kon.
