FIGURE 3.

Global data fitting of TTP incorporation and RNase H cleavage. Data for TTP incorporation (A) and concomitant RNase H cleavage (B) were analyzed by global data fitting. Two control experiments were performed to measure the RNase H activities of HIVRT against substrates before (C) or after (D) primer extension. Briefly, preformed RT·DNA/RNA complexes (175 nm HIVRT and 75 nm d25/r45 (C) or d26/r45 (D)) were rapidly mixed with 10 mm Mg²⁺ in the absence of any nucleotides. Each band was quantified individually so more information could be derived from the global fitting. Color codes are as follows. A, 25-nt primer, red; 26-nt product, green; 27-nt product, blue. B–D, 45-nt RNA template, red; cleavage at position −18 (38-nt), green; cleavage at position −17 (37-nt), blue; cleavage at position −16 (36-nt), yellow; cleavage at position −15 (35-nt), cyan. Note that because the data are displayed on a logarithmic timescale, the zero time point, which was included in the data fitting, is not shown. All the data were fit to a single model using KinTek Explorer to derive the rate constants summarized in Scheme 1 and Table 1. Smooth lines in each panel represent the best global fit of all four experiments.