Fig. 8.

Self-complementary TAR dimerization promotes very strong PKR activation. Native gel and activation assays were performed with scTAR (panels (a) and (b)) and pscTAR (panels (c) and (d)). (a) Conditions promoting self-complementary TAR (scTAR) monomer and dimer formation were optimized and analyzed on a native gel. Monomer (M) was prepared by renaturation of 1 µM RNA in 1×TE at 90 °C for 3 min, followed by incubation at room temperature for 10 min (heat condition 1) (Lane 1). Preparation of dimer (D) was attempted by renaturation of 20 µM RNA in 1×TE, 3.2 M NaCl using heat condition 1 (Lane 2), or incubating at 98 °C for 5 min, followed by slow cooling to ~40 °C over 1.5 h (heat condition 2) (Lane 3). Only the latter conditions gave dimer. (10% polyacrylamide native gel /0.5×TBE). (b) Activation assays using scTAR renatured at 0.8 µM in 1×TE for monomer with heat condition 1, or at 23 µM in 1×TE, 3.2 M NaCl for dimer with heat condition 2, followed by serial dilution to 0.01, 0.05, and 0.2 µM concentrations in 70 mM NaCl to maintain constant ionic conditions for these high-salt prepared RNAs. Gels are 10% SDS-PAGE, and PKR concentration was 0.8 µM. (c) Conditions promoting perfectly self-complementary TAR (pscTAR) monomer and dimer formation were optimized and analyzed on a native gel. Monomer and dimer were prepared by renaturation in 1×TE at 90 °C for 3 min, followed by incubation at room temperature for 10 min. Concentrations of pscTAR ranging from 0.2 to 20 µM were tested to determine optimal RNA conditions for monomer (M) or dimer (D) formation. (10% polyacrylamide native gel /0.5×TBE). (d) Activation assays using RNAs that were renatured in 1×TE at 0.4 µM for monomer or 20 µM for dimer, followed by serial dilution to 0.01, 0.05, and 0.2 µM concentrations. Gels are 10% SDS-PAGE, and PKR concentration was 0.8 µM. A bell-shaped dependence of activation on scTAR and pscTAR concentration is observed, wherein 0.05 µM dimer gives very strong activation of PKR, while scTAR and pscTAR monomers do not significantly activate PKR. 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 b and d).