Activation of PKR by Stem-Loop RNAs with Flanking SsRNA Tails

Abstract

Protein Kinase R (PKR) is a central component of the innate immunity antiviral pathway and is activated by double stranded RNA (dsRNA). PKR contains a C-terminal kinase domain and two tandem dsRNA binding motifs. In the accepted model for activation, binding of multiple PKR monomers to dsRNA enhances dimerization of the kinase domain. A minimum dsRNA length of 30 bp is required for binding two PKR monomers and eliciting strong enzymatic activation. However, short (15 bp) stem-loop RNAs containing flanking single stranded tails (ss-dsRNAs) are capable of activating PKR. Activation requires a 5'-triphosphate and the presence of both 5' and 3' ssRNA tails. The mechanism of PKR activation by ss-dsRNAs is not understood. Here, we have characterized the structural features of ss-dsRNAs that contribute to activation. We have designed a model ss-dsRNA PKR activator containing two single stranded tails of 15 nt and a 15 bp stem (5'-15-15-15-3') and made systematic truncations of the tail and stem regions. Analytical ultracentrifugation experiments combined with autophosphorylation assays were used to correlate RNA binding affinity with the ability to activate the kinase. Both tails are required for PKR activation; however, they can be truncated to produce either 10-15-5 or 5-15-10 without abolishing activation. Activity is retained upon reducing the stem to 10 bp but is lost upon further reduction to 5 bp. All of the ss-dsRNAs bind two PKR monomers in 75 mM NaCl. The loss of PKR activation is correlated with weaker PKR binding, consistent with a model where the autophosphorylation rates are proportional to the concentration of RNA species containing two PKR monomers. A 5'-triphosphate is required for activation. Although the binding affinity decreases upon removal of the 5'-triphosphate, the reduction is not sufficient to explain the loss of activation.