FIGURE 3.

Fe²⁺ selectively weakens IRE-RNA/IRP1 interactions. A, IRE-RNA binding to IRP was measured, ±Fe²⁺-O₂, as IRP1 fluorescence quenching in solution (constant protein concentration) as described in the legend to Fig. 2 (see “Experimental Procedures”). The IRE-RNAs used were FerH IRE-RNA, FerH ΔU⁶ IRE-RNA, and mt-aconitase IRE-RNA. Data were analyzed as previously described (45, 46). The K_d values are the averages of three titrations. Relative stabilities (1/K_d) of the IRE-RNA·IRP1 complexes are used in the graph, right. B, IRE-RNA binding to IRP ± Fe²⁺-O₂, was measured by electrophoretic mobility shift assay (constant RNA concentration). Anaerobic solutions of RNA and protein were prepared as in A; Fe²⁺ was 50 μm when present (see “Experimental Procedures”). A constant 0.1 μm RNA concentration was used with varying concentrations of protein; RNA was stained with ethidium bromide. Note the larger amount of IRP1 repressor required to bind (shift) an equivalent amount of mt-aconitase IRE-RNA compared with ferritin IRE-RNA ± Fe²⁺-O₂. The data are representative of two independent experiments and the significance of the effect of metal ions on the K_d values is p < 0.01.