PKR activation by ppp-ssRNA and heparin is inhibited by dsRNA, but not by short ppp-ssRNA. RNA-competition assays. (A) Competition for dsRNA-79-mediated PKR activation by short dsRNA and ssRNA. PKR activation by dsRNA-79 was assayed in the presence of increasing concentrations dsRNA-20 and ppp-ssRNA-20. The concentration of dsRNA-79 was held constant at 0.1 μM, and concentrations of dsRNA-20 and ppp-ssRNA-20 were 0.1, 1, 2.5, and 5 μM. (B) Competition for ppp-ssRNA-47-mediated PKR activation by short dsRNA and ssRNA. PKR activation was assayed as in A, with the concentration of ssRNA-47 held constant at 2.5 μM. (C) Competition for heparin-mediated PKR activation by short dsRNA and ssRNA. PKR activation was assayed as in A, with the concentration of heparin (average molecular weight of 6 kDa) held constant at 75 μg/mL, and concentrations of competitor RNAs at 0.1, 1, 2.5, 5, and 10 μM. For all panels, 10% SDS-PAGE gels are shown, with the position of phosphorylated PKR (p-PKR) indicated. The “no-RNA” and “no-competitor” lanes are included as negative and positive controls, respectively. Activation in the absence of added RNA was negligible. Phosphorylation activities are provided under the gels and were normalized to the 0.1 μM competitor lane for each set of competitors; activities were normalized in this manner because in some cases the presence of competitor at low concentrations appeared to stimulate the reaction.
