Figure 4.

Effects of the order of addition of FluB-ht (E), substrate (S), BXA (I), and MgCl₂on the endonuclease activity of WT and PA I38T FluB-ht.A, order of addition of reaction components in the absence of BXA has negligible effects on reaction progress for both WT (top) and PA I38T (bottom) FluB-ht. B, Preincubation E, I, and MgCl₂ is required for inhibition of WT (top) endonuclease activity. Inhibition of PA I38T (bottom) is not observed regardless of the order of addition the reaction components. Data represent at least three independent experiments. Error bars were omitted for clarity, Figure 4 with error bars is provided in the supplemental information (Fig. S2). All experiments were performed under the following final conditions: 100 nM 20-nt substrate, 30 mM Tris-pH 7.5, 5% DMSO, 25 mM NaCl, 75 nM BXA, 55 nM FluB-ht, 1.7 μM vRNA, and 5 mM MgCl₂.