Figure 6.

smFRET analysis of the 5′UTR dimer from the NL4.3 strain. (A) Bivariate histogram (ES plot) for the NL4.3-5′UTR²³⁸ dimer, formed in PI buffer at 1 mM Mg²⁺ for 1 h at 37°C and acquired at room temperature with the intra-UTR FRET assay. The histograms are normalized to the total number of data points included in each data set. The probability per histogram bin is displayed according to the indicated colormap, with red indicating the highest probability. Dotted lines indicate the thresholds used for identification of subpopulations of molecules, as defined in Fig. 5. The sizes of the subpopulations are summarized in Fig. 7. Also shown are univariate histograms displaying the distributions of FRET efficiency (plotted along the top horizontal axis) and stoichiometry (plotted along the right vertical axis). (B) Equivalent data, displayed in the same manner, obtained with the inter-UTR assay. (C) ES plot for NL4.3-5′UTR²³⁸ dimerized and imaged in PI buffer with 5 mM Mg²⁺ with the intra-UTR assay and (D) the same data obtained with the inter-UTR assay. (E) ES plot for NL4.3-5′UTR²³⁸ dimerized and PI buffer at 1 mM Mg²⁺ for 1 h at 57°C, followed by imaging at room temperature with the intra-UTR assay and (F) the same data obtained with the inter-UTR assay. (G) Intra-UTR FRET data for NL4.3-5′UTR²³⁸ dimerized in the presence of cognate NC^NL4.3 in PI buffer at 1 mM Mg²⁺ for 1 h at 37°C. (H) The same data obtained with the inter-UTR FRET assay. To see this figure in color, go online.