Charge density of divalent metal cations determines RNA stability - PubMed (original) (raw)

. 2007 Mar 7;129(9):2676-82.

doi: 10.1021/ja068027r. Epub 2007 Feb 13.

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Charge density of divalent metal cations determines RNA stability

Eda Koculi et al. J Am Chem Soc. 2007.

Abstract

RNA molecules are exquisitely sensitive to the properties of counterions. The folding equilibrium of the Tetrahymena ribozyme is measured by nondenaturing gel electrophoresis in the presence of divalent group IIA metal cations. The stability of the folded ribozyme increases with the charge density (zeta) of the cation. Similar scaling is found when the free energy of the RNA folded in small and large metal cations is measured by urea denaturation. Brownian dynamics simulations of a polyelectrolyte show that the experimental observations can be explained by nonspecific ion-RNA interactions in the absence of site-specific metal chelation. The experimental and simulation results establish that RNA stability is largely determined by a combination of counterion charge and the packing efficiency of condensed cations that depends on the excluded volume of the cations.

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Figures

Figure 1

Figure 1. Folding mechanism of Tetrahymena ribozyme

U, unfolded RNA in low ionic strength; I, misfolded intermediates, IN, native-like intermediates that can form in a counterion, N, native ribozyme that is formed in Mg2+. Adapted from Ref.

Figure 2

Figure 2. Folding of Tetrahymena ribozyme by group IIA metal ions

Folding reactions were in 50 mM Na-Hepes, pH 7.5, 1 mM EDTA (HE), 10% (v/v) glycerol, 0.01% xylene cyanol plus metal chloride at 30 °C. (a) Sample native 8% polyacrylamide gel. The ribozyme was folded in the concentrations of MgCl2, CaCl2, SrCl2 or BaCl2 shown above each lane. I is the misfolded RNA, N is the native RNA. (b) Fraction of native RNA (_f_N) versus cation concentration. Curves represent the best fit to the Hill equation, with the parameters given in Table 1. (c) Folding free energy of the Tetrahymena L-21Sca ribozyme (ΔGUN(Me)) vs. cation charge density (ζ). ΔGUN(Me) was taken from the cooperativity of the transitions in (b). Error bars are the variation in two independent trials. For each ion, ζ was calculated from the volume of a sphere with radius equal to the length of the metal-oxygen bond.

Figure 3

Figure 3. Urea denaturation of the Tetrahymena ribozyme

(a) Ribozyme RNA folded in HE plus 3 mM MgCl2, CaCl2, SrCl2 or BaCl2 at 30°C was denatured with the addition of 0 − 5 M urea. The fraction of folded RNA, _f_N, was measured by native PAGE and fit to the linear extrapolation model as described in Methods and Table 2. (c) Correlation of the folding free energy in water extrapolated from the urea denaturation curves (ΔGUN(urea)) with ζ.

Figure 4

Figure 4. Relative gel mobility of ribozyme decreases with metal ion size

(a) Native 8% PAGE of the unfolded (U) or folded (F) Tetrahymena ribozyme, in running buffer containing 3 mM metal chloride (see Methods). The unfolded ribozyme samples were in HE buffer, the folded RNA was in HE plus 1.4 mM MgCl2, 1.4 mM CaCl2 or 6 mM SrCl2, respectively. ΦX, 5’-32P-labeled ΦX/Hae III DNA. (b) Relative mobility of the folded ribozyme compared to the 271 or 310 bp DNA, as a function of counterion charge density (ζ). Similar results were obtained in two trials.

Figure 5

Figure 5. Temperature dependence of folding in group IIA metals

ΔGUN(Me) was measured by titration with MeCl2 at 30°C, 45°C and 60°C as in Figure 2. Curves represent the best fit line. Error bars represent the variation in two or more trials.

Figure 6

Figure 6. Simulation of polyelectrolyte collapse by small and large metal ions

(a) The collapse dynamics of a model polyelectrolyte (N = 120) in divalent counterions of various sizes is shown as the change in the radius of gyration (Rg) with time. Each time trace is averaged over eight individual trajectories to minimize the statistical errors. (b) Internal energy of the collapsed state of the polyelectrolyte, for four different counterions. Inset: Rg of the collapsed polyelectrolyte as a function of the hydrodynamic radius of the counterion (Rion).

Figure 7

Figure 7. Ion-monomer distance distribution and correlations between condensed ions

(a) Radial distribution of the distances between the divalent ions and a model polyelectrolyte. (b) Pair correlation between ions that are condensed around the model polyelectrolyte. The correlation effects are reflected in the packing of the divalent around the compact state. (c) Compact globules in ions the size of Mg2+ (Rion = 2.07 Å) and Ba2+ (Rion = 2.99 Å). The volume of the globule in Ba2+ is nearly 30% larger than the one in Mg2+.

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