FIGURE 5.

Analysis by time-resolved FRET shows that the Kt-7 RNA contains two populations of conformations. Donor-acceptor (D-A) distance distributions were analyzed using the same fluorescein-Cy3 end-labeled species analyzed by steady-state FRET (Fig. 4 ▶), using frequency-domain phase fluorometry. (A) Plots of phase shift and demodulation of the fluorescein emission as a function of the presence of 1 nM (•) and 50 mM (□) magnesium ions as a function of the modulation frequency. The best fits to the experimental data using one or two Gaussian distributions of D-A lengths are shown by the broken and solid lines, respectively. Detailed analysis of the distribution of residuals and Bootstrap analysis shows that the fits using two D-A length distributions are superior. Both sets of data were fitted globally to give the distributions shown in B and C, with mean D-A distances of 23 and 83 Å. (B) Two-Gaussian D-A length distributions [P(R) as a function of distance R] calculated from the fit to the data obtained in 1 nM magnesium ions. The major species (65%) corresponds to the longer donor-acceptor distance. The integrated areas under the curves total 100%. (C) Two-Gaussian D-A length distributions calculated from the fit to the data obtained in 50 mM magnesium ions. The major species (70%) now corresponds to the shorter D-A distance. (D,E) Comparison of the phase residuals when one (D) or two (E) Gaussian distributions of distance are applied to the data acquired at low Mg²⁺ concentration. The resulting χ² values are 23.2 and 20.6, respectively. (F,G) Phase residuals when one (F) or two (G) Gaussian distributions of distance are applied to the data acquired at high Mg²⁺ concentration. The resulting χ² values are 62.6 and 36.8, respectively. (H) Bootstrap error analysis of the probable distance and half-width for the shorter distance shows the scatter of values about the best fit from 800 cycles.