Inhibition of 3Dpol by inhibitors is non-competitive with respect to RNA and NTPs.

<p>(A) Effect of inhibitors on binding of 3Dpol to template-primer (T/P). Binding of 3Dpol to fluorescently labeled T/P (T/P*) was assessed by determining the amount of 3Dpol-T/P* covalent complex formed by UV-mediated cross-linking in the presence of 20 µM of inhibitors. For these experiments, 2 µg 3Dpol was incubated with 100 nM T/P (poly-rA/5′-Cy3-dT₁₈) in a buffer containing 50 mM Tris-HCl pH 7.8, 1 mM DTT and 5 mM MgCl₂ at 4°C for 10 minutes. Cross-linking was carried out by exposing the mixture to UV for 3 minutes as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015049#s4" target="_blank">Materials and Methods</a>. The radiograph at the top shows equivalent amounts of 3Dpol-T/P adduct in the absence and presence of the inhibitors. The middle and bottom figures in panel A show that the amounts of poly-rA/5′-Cy3-dT₁₈ and protein, respectively are the same in all cross-linking experiments. (B) and (C) Non-competitive inhibition profile of FMDV 3Dpol by 5D9 under steady state conditions. Kinetic experiments with 3Dpol (1 µg/0.1 ml reaction volume) were conducted in 96-well plates using the luciferase-based assay (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015049#s4" target="_blank">methods</a>) in the presence of increasing concentrations of 5D9 (0 to 32 µM), varying either UTP substrate (1.6-102.4 µM) (panel B) or poly-rA/dT₁₈ (0.1 to 6.4 µM) (panel C). In both cases the X-axis intercepts (-1/K_m for the UTP or poly-rA/dT₁₈ substrates) are not affected by the inhibitor concentrations, which is the hallmark of non-competitive inhibition. Indicated values are the means from at least three independent experiments.