Figure 6.

RHA does not affect RT's binding and kinetic properties of single-nucleotide incorporation. A, determination of RNA template binding affinity (K_d,RNA) of RT in the absence and presence of RHA under pre–steady-state conditions. Active-site titration of RT in the presence and absence of RHA was carried out by preincubating RT (50 nm) in the absence (■) and presence of RHA (50 nm) (▴) with varying concentrations of P/T complex (40, 80, 160, 320, and 640 nm) followed by the addition of dTTP (100 μm) and MgCl₂ (2.5 mm). B, determination of RT's dissociation rate (k_off) from the P/T complex before nucleotide incorporation. Under steady-state conditions, 30 nm RT was incubated with 30 nm P/T in the presence and absence of RHA. The solutions were then mixed with dsDNA trap at varying time points and initiated with dTTP in which reactions were run for 10 s. RT's dissociation rate (k_off) was determined by fitting to a single exponential decay equation. C, comparison of observed catalytic rate (k_obs) of single-nucleotide incorporation under steady-state conditions. Reactions contained 5′-Cy3–labeled DNA primer annealed to the 3′-UTR (333 nm) and only dTTP (333 μm), which terminates the RT reaction at the +1 nt position. The k_obs was obtained by the quantification of the DNA primer extension products and nonlinear square fitting of the data. The average results of three independent experiments are shown with standard deviation (±).