Magnesium dependence and equilibrium of the Escherichia coli ribosomal subunit association

The well-known, magnesium ion-dependent, reversible association of Escherichia coli ribosomal subunits: 30 s + 50 s ⇌ 70 s has been quantitatively studied with a simple light-scattering approach which appears to be the most convenient means for the assay of this reaction in both directions. The results with this technique correlated exactly with those obtained by zone sedimentation. The reaction has been found to be a true equilibrium that is dependent upon both the concentrations of ribosomes and Mg 2+. Expressions have been formulated to describe the equilibrium, and the equilibrium constant and the number of Mg 2+ involved has been evaluated. The critical value of the latter is small relative to the total Mg 2+ bound per ribosome. Increased temperature enhances dissociation of the 70 s particle and leads to a lowering in the critical number of Mg 2+. From the temperature and equilibrium studies the following thermodynamic constants have been determined: ΔG° = −35·3 kcal./mole (at 25°C), ΔH° = − 70 kcal./mole and ΔS° = 120 e.u. (all determined in 50 mu-Tris buffer (pH 7·8) and 50 mM-KCl). The enthalpic change is the dominant term and the magnitude of the parameters are suggestive of a conformational change in structure in the course of the reaction. A variety of cations (potassium, calcium and polyamines) which have previously been shown to influence the reaction were studied. Potassium, similar to increased temperature, favors dissociation of the 70 s particle, with decreases in the magnitude of the thermodynamic parameters. Polyamines, on the other hand, favor association of the two subunits, with spermidine being 100-fold more effective on a molar basis than putrescine. Both potassium and the polyamines reduced the critical number of Mg 2+ as reflected in a broadening of the Mg 2+ concentration range over which the transition takes place. This is due to either a direct competition of the cations with Mg 2+, or an indirect induced conformational change in the ribosome. Calcium could be substituted for Mg 2+, but was much less effective in enhancing association. The dissociation pattern of native 70 s ribosomes is multiphasic and differs from the dissociation of purified particles. However, reassociation of these subunits was identical to the pattern with purified subunits.