Fig. 2.

FRET-related histograms for Pol I(KF) complexes. (A–C) FRET-related histograms for (A) unliganded R₅₅₀G₇₄₄ Pol I(KF), (B) the Pol-DNA binary complex, and (C) the Pol-DNA-dNTP ternary complex (with a correct dNTP, forming an A-dTTP pair). The numbers of bursts in A, B, and C are comparable (2731, 2908, and 2598 events, respectively). The binary complex was formed by adding 100 nM DNA to Pol I(KF); the ternary complex was formed by adding 100 nM DNA and 10 μM dTTP to Pol I(KF). Each sample is described by two histograms: The lower shows a two-dimensional histogram of probe stoichiometry, S, vs. apparent FRET efficiency, E^∗, for each diffusing molecule that contains both G and R fluorophores (i.e., molecules with 0.6 < S < 0.9; see SI Materials and Methods and refs. 14, 31). The upper panel shows a one-dimensional E^∗ histogram (80 bins) of the molecules in the lower panel. In (B) and (C), the E^∗ distributions were fitted to double-Gaussian distributions (black solid lines, sum of Gaussians; dashed lines, individual Gaussians) using an iterative process (see SI Materials and Methods). The percentage of the population in each Gaussian (after correction for the contaminating G₅₅₀R₇₄₄ protein) is indicated. The two dashed vertical lines mark the mean E^∗ values of the main subpopulations in the binary (open) and ternary (closed) complexes. For the unliganded Pol I(KF) in (A), the red line shows the best fit by a single Gaussian; the black dashed lines show a fit to a constrained double-Gaussian distribution with the means and standard-deviations of the Gaussian peaks set to equal those of the open (〈E^∗〉 = 0.5, σ_SN = 0.054) and closed (〈E^∗〉 = 0.7, σ_SN = 0.060) complexes determined from the data in (B) and (C); neither of these strategies provide a good fit to the experimental data (see text). The residuals of all fits are in Fig. S5.