Single-molecule FRET analysis of the three-way IRES junction with helix D of the natural sequence. (A) The sequence of the junction studied. This is identical to that of the simplified junction except for the restoration of the base pairing found in the natural junction. However, the terminal loop was removed and the base pairing extended to form a stable helix with a 5′-terminus for fluorophore attachment. The junction is formally depicted in the 3HS4 secondary structure. (B) Population distributions of FRET efficiencies for Cy3–Cy5-labeled vectors encapsulated in phospholipid vesicles in 10 mM Tris-HCl (pH 8.1), 50 mM NaCl, 10 mM MgCl2, and imaging buffer measured at 100-msec time resolution. The total number of molecules included were 1693 and 860 for the CD and CE vectors, respectively. (C) A representative 1-sec time record for the CD vector at 8-msec resolution (left). Four seconds of data were used as input into a cross-correlation analysis (right). These data show no evidence of structural transitions at this time resolution.
