Fig. 4.

RNA hairpin dimerization promotes PKR activation. Activation and inhibition assays (10% SDS-PAGE) were performed at various concentrations of PKR, as indicated to the right of each gel. (a) Activation assays with native gel-purified TARwt monomer and dimer in the presence of 5 µM PKR. Higher PKR concentration (5 µM) promotes activation by TARwt dimer, with just weak activation by monomer. (b) Activation assays with native gel-purified A34U:U37A monomer and dimer in the presence of 0.8 µM PKR. (c) Activation assays with native gel-purified A34U monomer, U37A monomer, and [A34U + U37A] dimer in the presence of 0.8 µM PKR. For both panels (b) and (c), lower PKR concentration (0.8 µM) achieves activation by dimer with (at most) weak activation by monomer. (d) Inhibition assay with native gel-purified TARwt TAR monomer and dimer. All lanes have 0.1 µM PKR and 0.01 µM 79 bp dsRNA, which is a potent activator of PKR. Increasing the concentration of TARwt monomer from 0.01 to 1.2 µM inhibits activation ~1.5-fold, while the same increases in wtTAR dimer concentration inhibits activation ~2-fold. For all panels, a no-RNA lane (−) is provided, and phosphorylation activities are normalized to 0.01 µM 79 bp RNA. In order to provide RNA-dependent activation values, the no-RNA lane was subtracted from each lane. These background-subtracted values are provided, as are RNA-dependent fold-effects for dimer over monomer (panels a and b) or for inhibition relative to 79 bp RNA (panel d).