Analysis of the structural flexibility of nucleotides in the IRES three-way junction using in-line probing. Radioactively [5′-32P]-labeled RNA was incubated in 50 mM Tris-HCl (pH 8.3), 100 mM KCl, and 20 mM MgCl2 for various times at 21°C, and analyzed by denaturing gel electrophoresis and phosphorimaging. The junction was studied as a single strand by placing terminal loops onto two helices. In order to maximize the resolution of the sequences of interest, two constructs were studied, each with a single 5′-terminus in either helix D (A) or helix C (B). Bands were assigned by reference to cleavage by ribonuclease T1 (A, lanes 1,8; B, lane 3) and a hydroxide cleavage ladder (A, lanes 2,7; B, lane 2). The junction with open helix D was subjected to in-line probing for 5 min, 12, 24, and 48 h (A, lanes 3–6, respectively), and the open C arm form to a 48-h incubation (B, lane 1). The nucleotides were numbered according to Honda et al. (1999).
