Activation of PKR by Stem Loop RNAs Requires Flanking ssRNA Arms

Protein kinase R (PKR) is a key component of the interferon-induced viral response pathway. PKR contains an N-terminal double-stranded RNA (dsRNA) binding domain and a C-terminal kinase domain connected by a ∼90-residue linker. Upon binding dsRNA, PKR undergoes autophosphorylation and kinase activation. Activated PKR then phosphorylates the alpha subunit of the eukaryotic initiation growth factor 2 and thus inhibits protein synthesis in virally infected cells. A minimum of 30 bp of dsRNA is required to bind two PKRs, leading to dimerization and activation. Stem-loops are common RNA structural motifs. Recently, it was reported that short stem-loops containing 16 bp and ssRNA flanking arms are also able to activate PKR and that activation requires a 5’-triphosphate. However, the activation mechanism is not known. We have prepared RNAs containing a 15 bp stem-loop and various ssRNA flanking arms and characterized activation and binding of PKR. Activation by these RNAs requires ssRNA arms on both the 5’ and 3’ sides but it is independent of the presence of a 5’-triphosphate. Sedimentation velocity measurements indicate that a single PKR binds to the activating stem-loop RNA at 200 mM NaCl with Kd = 309 nM. PKR binding affinity is not strongly affected by removal of either of the ssRNA arms or the 5’-triphosphate. At lower [NaCl] (75 mM), each of the stem-loop RNAs can bind two monomers of PKR. However, only the activating RNA exhibits a positive binding cooperativity. We propose that activation by this RNA is due to enhanced population of the species containing two bound PKRs as a result of cooperative binding of the second PKR monomer.