The RNA-activated protein kinase, PKR, is induced by interferon and plays a pivotal role in the innate immunity response to viral infection. PKR is synthesized in a latent state, but upon binding activating RNAs it undergoes autophosphorylation reactions that activate the kinase. Phosphorylation of the eukaryotic initiation factor eIF2a by PKR inhibits viral protein synthesis. PKR is also activated by cytokines, growth factor withdrawal and stress signals and participates in pathways regulating stress response, cellular growth and proliferation, nutrient signaling and metabolism. The broad objective of our research program is to define the molecular mechanisms that regulate the activity of PKR using quantitative biophysical and structural methods. In Aim I we will define the interactions of PKR with complex RNA inhibitors and activators. Specifically, we will identify the structural features within the Adenovirus VAI RNA that mediate high-affinity PKR binding without activating the kinase, determine the mechanism of activation of PKR by novel single stranded-double stranded RNAs and correlate PKR dimerization and activation with the length and flexibility of a linker region in model RNAs. PKR represents an attractive target for development of novel antiviral therapies to enhance the innate immune response and in Aim 2 we will use a nonradioactive assay to screen libraries of drug-like compounds for novel PKR activators. In Aim 3, small angle scattering methods will be used to determine the conformation of ordered forms of PKR and to develop structural model of complexes of PKR with RNA inhibitors and activators. These studies will provide the foundation for the design of therapeutic agents that target PKR for the treatment of viral infections and cancer.