Dissecting electrostatic screening, specific ion binding, and ligand binding in an energetic model for glycine riboswitch folding

Abstract

Riboswitches are gene-regulating RNAs that are usually found in the 5'-untranslated regions of messenger RNA. As the sugar-phosphate backbone of RNA is highly negatively charged, the folding and ligand-binding interactions of riboswitches are strongly dependent on the presence of cations. Using small angle X-ray scattering (SAXS) and hydroxyl radical footprinting, we examined the cation dependence of the different folding stages of the glycine-binding riboswitch from Vibrio cholerae. We found that the partial folding of the tandem aptamer of this riboswitch in the absence of glycine is supported by all tested mono- and divalent ions, suggesting that this transition is mediated by nonspecific electrostatic screening. Poisson-Boltzmann calculations using SAXS-derived low-resolution structural models allowed us to perform an energetic dissection of this process. The results showed that a model with a constant favorable contribution to folding that is opposed by an unfavorable electrostatic term that varies with ion concentration and valency provides a reasonable quantitative description of the observed folding behavior. Glycine binding, on the other hand, requires specific divalent ions binding based on the observation that Mg(2+), Ca(2+), and Mn(2+) facilitated glycine binding, whereas other divalent cations did not. The results provide a case study of how ion-dependent electrostatic relaxation, specific ion binding, and ligand binding can be coupled to shape the energetic landscape of a riboswitch and can begin to be quantitatively dissected.

FIGURE 1.

(A) Secondary structure of the VCI-II riboswitch tandem aptamer from Mandal et al. (2004) (B) Three-state thermodynamic model for the VCI-II aptamer with the unfolded (U), high salt (M), and glycine-bound conformations (B) summarizing the observed ion and ligand dependencies.

FIGURE 2.

SAXS data for the VCI-II aptamer in the presence of different ions in the absence and presence of glycine. VCI-II SAXS profiles in the presence of 2 M Na⁺ (A), 2 M K⁺ (B), 20 mM Ca²⁺ (C), 20 mM Mn²⁺ (D), 20 mM Sr²⁺ (E), 20 mM Ba²⁺ (F), and 20 mM Zn²⁺ (G), in the absence (green, solid lines) and presence (red, solid lines) of 20 mM glycine. For comparison, profiles in 50 mM Na-MOPS buffer only (blue, dashed lines), in the presence of 20 mM Mg²⁺ (green, dashed lines), and in the presence of 20 mM Mg²⁺ and 20 mM glycine (red, dashed lines) are shown. Data are shown in Kratky representation (q^2.I as a function of q, where I is the scattering intensity and q is defined as q = 4π sin(θ)/λ, with λ being the X-ray wavelength and 2θ the total scattering angle), which is particularly sensitive to conformational changes (Lipfert et al. 2009). The lower signal-to-noise at high q (which is emphasized by the Kratky representation) noticeable in the profiles for 2 M Na⁺ and K⁺ is due to X-ray absorption and reduced scattering contrast at the high salt concentrations.

FIGURE 3.

Comparison of normalized band intensities from hydroxyl radical footprinting of 5′-radiolabeled VCI-II RNA. (A) Profiles in the absence of glycine under different ionic conditions: 100 mM Mg²⁺ (green); 100 mM Ca²⁺ (orange); 100 mM Sr²⁺ (brown); and 2 M Na⁺ (black). (B) Profiles in the presence of 10 mM glycine under different ionic conditions: 100 mM Mg²⁺ (red); 100 mM Ca²⁺ (orange); 100 mM Sr²⁺ (brown); and 2 M Na⁺ (black). (C) Profiles in 2 M Na⁺ in the absence (black, dashed) and presence (black, solid) of 10 mM glycine. (D) Profiles in 100 mM Sr²⁺ in the absence (brown, dashed) and presence (brown, solid) of 10 mM glycine. Error bars from independent gels are omitted for clarity. Regions with significant differences in the protection patterns between different ion species are shaded in B).

FIGURE 4.

SAXS data as a function of Na⁺ concentration in the absence of glycine. (A) Scattering profiles of the VCI-II aptamer in Kratky representation (q^2.I as a function of q) in the presence of 50, 100, 150, 300, 550, 800, 1050, and 2050 mM Na⁺, color coded from blue (low Na⁺) to red (high Na⁺). (B) VCI-II radii of gyration (black symbols) obtained from Guinier fits to scattering data (left axis R_g² in Ų, right axis R_g in Å) as a function of total Na⁺ concentration and the Hill fit to the data (black solid line). The errors are obtained from Guinier fits with slightly different fitting ranges. (Inset) Fractional population of the M state (f_M) from two-state projections of the SAXS scattering profiles and the corresponding Hill fit to the data (solid lines).

FIGURE 5.

Low-resolution three-dimensional models of the VCI-II aptamer in the U, M, and B conformations. Models were obtained from ab initio reconstructions from SAXS data (see Materials and Methods). The scale bar corresponds to 20 Å, the diameter of an RNA helix.

FIGURE 6.

The U-to-M transition as a function of Na⁺ and Mg²⁺ concentrations monitored by SAXS compared with Poisson–Boltzmann (PB) predictions of the electrostatic effects from the ion atmosphere. (A) Fractional occupancy of the M state as a function of Na⁺ concentration from SAXS (R_g fitting, ○) and prediction from PB theory (solid line). (B) Fractional occupancy of the M state as a function of Mg²⁺ concentration from SAXS (R_g fitting, ⋄) and prediction from PB theory (solid line).

FIGURE 7.

Solvent exposure of the VCI-II tandem aptamer probed by hydroxyl radical cleavage as a function of Mg²⁺ concentration in the absence (A) and presence (B) of 10 mM glycine, both with a 2 M NaCl background. Data are for residues 47–150 and were normalized such that the amount of cleavage at 0 mM Mg²⁺ corresponds to zero (for each of the titration sets). Blue regions show increasing protections from cleavage, and red regions show increasing cleavage. In the presence of glycine (B), protections increase for certain regions, while no protections were increased when no glycine was added (A). This observation gives further evidence that the M state is populated in 2 M NaCl. For comparison, relative protections determined from a glycine titration in 10 mM Mg²⁺ background are shown (far right). The data in this reference lane are normalized to 10 mM Mg²⁺ and no glycine, and are taken from Lipfert et al. (2007b). This glycine titration in the 10 mM Mg²⁺ background probes the M-to-B transition, and hence, can be directly compared with both A and B, where the M state is populated in 2 M NaCl. This same M-to-B transition is observed in B.

FIGURE 8.

Schematic phase diagrams for the VCI-II aptamer showing the population of the unfolded (U, blue), high salt (M, green), and glycine-bound conformations (B, red) summarizing the observed ion and ligand dependencies. Phase diagrams are shown for the absence of glycine (A) and for the presence of a high concentration of glycine (B). For divalent ions that do not support glycine binding (Sr²⁺, Ba²⁺, and Zn²⁺) the phase diagram in the presence of glycine is identical to that in the absence of glycine. Details of how the diagrams were constructed are given in the “Construction of phase diagrams” section in Materials and Methods.