Steady-state FRET analysis of the conformation and folding of the IRES junction. For these experiments, we use junctions with arms of 12 bp in length (see example in Fig. 1C), in which two arms are terminally 5′-labeled with fluorescein (donor) and Cy3 (acceptor), and measure the energy transfer between them. We have measured FRET efficiency (EFRET) for six vectors, corresponding to different end-to-end distances. The patterns of efficiencies correspond well to the global structures deduced from gel electrophoresis (Fig. 2). (A,B) Histograms of the FRET efficiencies for the six vectors for the IRES junction in 0 mM (A) and 10 mM (B) Mg2+. (C) FRET efficiencies for the 4H junction in 10 mM Mg2+.
