Analysis of metal ion-induced folding of the IRES three-way junction by steady-state FRET measurements. The junction shown in Figure 1C was prepared with fluorescein (donor) and Cy3 (acceptor) fluorophores attached to the 5′-termini of selected helical arms. These vectors are named by the labeled arms, in the order donor–acceptor, so that the CD vector has fluorescein attached to helix C and Cy3 to helix D. FRET efficiency was measured using the acceptor normalization procedure as a function of Mg2+ ion concentration in 90 mM Tris-borate (pH 8.3), and plotted for the three vectors CD (■), DE (○), and CE (●). The data have been fitted to a two-state model of ion-induced folding (—).
