FIGURE 2.

(A) Diagram of telomerase RNA construct used in the current study. Telomerase RNA was labeled at U139 with a donor fluorophore (Cy3) and at U10 with an acceptor fluorophore (Cy5) for smFRET studies. The RNA construct was deposited on a quartz slide for TIRF microscopy by means of a 5′ extension designed to hybridize with a biotinylated DNA handle. The biotinylated handle was immobilized on the quartz slide via a biotin-streptavidin linkage. (B) Histogram demonstrating the FRET distribution of dye-labeled RNA molecules in the absence of p65. The distribution is centered at 0.26 FRET. (C–F) Histograms of the FRET distribution of dye-labeled RNA molecules in the presence of 10 nM p65^FL (C), 32 nM p65^ΔN (D), 64 nM p65^ΔC (E), or 750 nM p65^CTD (F). FRET is defined as I_A/(I_A + I_D), where I_A is the intensity of the acceptor dye and I_D is the intensity of the donor dye. The protein concentrations used were determined by EMSA to have a large fraction of p65-RNA complexes (see Fig. 1C).