Role of the central junction in folding topology of the protein-free human U2–U6 snRNA complex

Calculation of a decay curve for the three-helix conformer (cyan) by subtraction of the experimentally obtained decay curves of fluorescein-labeled 4HJΔL1 (orange) from that of WTΔL (green). (A) Curves without added Mg²⁺ and (B) curves with added Mg²⁺ (40 mM). Concentration of dye-labeled RNA was 150 nM. trFRET measurements were recorded on a timescale of 20 nsec to a total of 10,000 counts in the peak. The decay curve of fluorescein on 4HJΔL1 and WTΔL were fit by a monoexponential and biexponential curve, respectively, with an excellent R² (R² > 0.999). The calculated decay curve for the three-helix conformer in each case was fitted with a monoexponential curve yielding a less perfect fit (R² value ∼0.974 and 0.911 without and with Mg²⁺, respectively), suggesting a mix of several three-helix conformers (see Results for details).