title: Activation of PKR by short stem-loop RNAs containing single-stranded arms.
abstract: Protein kinase R (PKR) is a central component of the innate immunity antiviral pathway and is activated by dsRNA. PKR contains a C-terminal kinase domain and two tandem dsRNA binding domains. In the canonical activation model, binding of multiple PKR monomers to dsRNA enhances dimerization of the kinase domain, leading to enzymatic activation. A minimal dsRNA of 30 bp is required for activation. However, short (∼15 bp) stem-loop RNAs containing flanking single-stranded tails (ss-dsRNAs) are capable of activating PKR. Activation was reported to require a 5'-triphosphate. Here, we characterize the structural features of ss-dsRNAs that contribute to activation. We have designed a model ss-dsRNA containing 15-nt single-stranded tails and a 15-bp stem and made systematic truncations of the tail and stem regions. Autophosphorylation assays and analytical ultracentrifugation experiments were used to correlate activation and binding affinity. PKR activation requires both 5'- and 3'-single-stranded tails but the triphosphate is dispensable. Activation potency and binding affinity decrease as the ssRNA tails are truncated and activation is abolished in cases where the binding affinity is strongly reduced. These results indicate that the single-stranded regions bind to PKR and support a model where ss-dsRNA induced dimerization is required but not sufficient to activate the kinase. The length of the duplex regions in several natural RNA activators of PKR is below the minimum of 30 bp required for activation and similar interactions with single-stranded regions may contribute to PKR activation in these cases.
authors.affiliation: Department of Molecular and Cell Biology, University of Connecticut, Storrs, Connecticut 06269, USA.
authors.forename: Christopher B
authors.initials: CB
authors.lastname: Mayo
authors.affiliation: Department of Molecular and Cell Biology, University of Connecticut, Storrs, Connecticut 06269, USA.
authors.forename: C Jason
authors.initials: CJ
authors.lastname: Wong
authors.affiliation: Department of Molecular and Cell Biology, University of Connecticut, Storrs, Connecticut 06269, USA.
authors.forename: Prisma E
authors.initials: PE
authors.lastname: Lopez
authors.affiliation: National Analytical Ultracentrifugation Facility, University of Connecticut, Storrs, Connecticut 06269, USA.
authors.forename: Jeffrey W
authors.initials: JW
authors.lastname: Lary
authors.affiliation: Department of Molecular and Cell Biology, University of Connecticut, Storrs, Connecticut 06269, USA Department of Chemistry, University of Connecticut, Storrs, Connecticut 06269, USA National Analytical Ultracentrifugation Facility, University of Connecticut, Storrs, Connecticut 06269, USA.
authors.forename: James L
authors.identifier: 0000-0002-9028-8364
authors.initials: JL
authors.lastname: Cole
mesh_terms: D019281:Dimerization; D004789:Enzyme Activation; D012330:RNA, Double-Stranded / Q000737:chemistry / Q000378:metabolism*; D019892:eIF-2 Kinase / Q000378:metabolism*
chemical_list: D012330:RNA, Double-Stranded; D019892:eIF-2 Kinase
keywords: PKR; dsRNA; innate immunity; protein kinase