Figure 1. Titration of a ‐labeled RNA with the RRM domain of hnRNP G protein. Here, the RNA is present at high concentration, for estimation of fractional binding activity. The 187 nt RNA probe (3 × 10 ⁻⁷ M) was titrated with 0–8 × 10 ⁻⁶ M hnRNP G protein (total concentration is the sum of active and inactive protein forms). Binding buffer containing 30 mM Tris (pH 7.8), 20 mM KCl, 2 mM MgCl ₂, 4 mM reduced glutathione, 4% glycerol. Volumes and nominal concentrations of components in these reactions are given in Table 3. Incubation was for 20 min at 30 °C. The reactions were transferred to ice, heparin was added to a final concentration of 0.05 mg ml ⁻¹, and 1/10 volume of gel loading buffer was added with gentle mixing. Samples were applied to a 6% polyacrylamide gel cast and run in TBE buffer (45 mM Tris–borate, 2.5 mM EDTA (pH 8.3 at 20 °C)). Electrophoresis was carried out at room temperature for 30 min at 10 V cm ⁻¹. Band designations: B = bound RNA–protein complexes; F = free RNA. Note the presence of a minor free RNA species (labeled “a”) and three complexes (two that nearly comigrate labeled “b.” and one additional species labeled “c”).

Figure 2. Estimation of fractional binding activity. The concentration of RNA bound is shown as a function of the concentration of hnRNP G protein added. Binding data are from integration of band intensities for the samples shown in Figure 1 interpreting all mobility‐shifted species as RNA–protein complexes and all unshifted species as free RNA. The protein concentrations shown are for total protein (active + inactive) in each sample. The solid line is a linear fit to the first 10 points in the concentration series, giving a slope of 0.02 ± 0.0004. This corresponds to ∼2% binding activity if binding stoichiometry is 1 : 1.

Figure 3. Titration of a ‐labeled RNA with the RRM domain of hnRNP G protein for determination of K _d. Here, the 187 nt RNA probe, present at low concentration (3 × 10 ⁻¹¹ M), was combined with 0–1.6 × 10 ⁻⁷ M binding‐active hnRNP G protein. The binding buffer contained 30 mM Tris (pH 7.8), 20 mM KCl, 2 mM MgCl ₂, 4 mM reduced glutathione, 4% glycerol. Volumes and nominal concentrations of components in these reactions are given in Table 2. Incubation was for 20 min at 30 °C. The reactions were transferred to ice, heparin was added to a final concentration of 0.05 mg ml ⁻¹, and 1/10 volume of gel loading buffer was added with gentle mixing. Samples were applied to a 6% polyacrylamide gel cast and run in TBE buffer (45 mM Tris–borate, 2.5 mM EDTA (pH 8.3 at 20 °C)). Electrophoresis was carried out at room temperature for 30 min at 10 V cm ⁻¹. Band designations: B = bound RNA–protein complexes; F = free RNA.

Figure 4. Estimation of K _d. (a) Fraction of RNA bound plotted as a function of log ₁₀[protein]. Binding data are from integration of band intensities for the samples shown in Figure 3, interpreting all mobility‐shifted species as RNA–protein complexes and all un‐shifted species as free RNA. The protein concentrations shown are for active protein present in each sample (i.e., 2% of total protein, as indicated by Figures 1 and 2). The solid curve is a least‐squares fit to Y = F _c × K(10 ^log[P])/(1 + K(10 ^log[P])), where Y is the fraction of RNA bound (Y = [P·R]/([R] + [P·R])); F _c is the fraction of RNA that is competent in binding, K is the association constant (K = 1/K _d), and [P] is the input protein concentration. If [P] is taken to be equal to the free protein concentration (justified by the fact that [RNA] ≪ [P] in this experiment), this fit returns K _d = 2.75 ± 0.16 × 10 ⁻⁸ M and F _c = 0.845; (b) Fraction of RNA bound plotted as a function of [active protein]. The solid curve is a fit to the quadratic form of the binding equation (Equation 2 in the text), returning K _d = 2.67 ± 0.38 × 10 ⁻⁸ M and F _c = 0.84.

Figure 5. Binding competition assay. Titration of a specific lac repressor–operator complex (B) with sheared calf thymus DNA. Transfer of repressor to the competitor produces the free lac operator DNA (F). Reactions were carried out in 10 mM Tris (pH 8.0), 1 mM EDTA, 150 mM KCl, 0.1 mg bovine serum albumin ml ⁻¹. All samples contained 3.3 × 10 ⁻¹¹ M lac operator DNA and 4.3 × 10 ⁻¹¹ M lac repressor. Samples 2 to 10 contained competing DNA at concentrations increasing from 4 × 10 ⁻⁷ M base pairs (sample 2) to 1 × 10 ⁻² M base pairs (sample 10).