The RNA-dependent protein kinase (PKR) is a key component of the interferon-mediated antiviral response. Induced by interferon, PKR phosphorylates and blocks the function of the translation initiation factor eIF-2 in virally infected cells, inhibiting the synthesis of viral proteins. PKR also has properties of a tumor suppressor, which are believed to arise from the activation of PKR by currently unidentified RNA structures in mRNAs encoding proteins involved in growth control. The enzyme is activated in vitro by binding to double stranded RNA (dsRNA) greater than 30 bp in length. However, recent discoveries of PKR-activating structures present in human mRNA and viral genomic RNA that do not have 30 bp duplex regions, indicate that RNA structural motifs more complex than simple, perfectly matched duplexes can activate the enzyme. To understand fully how specific RNA sequences can function to activate PKR and ultimately control protein synthesis and cell growth, we must identify RNA structures capable of binding to and activating the enzyme and fully characterize the binding mechanism. The research proposed here is directed at identifying PKR-activating structural motifs by selecting activating RNAs from sequence random RNA libraries and characterizing the selected molecules by secondary structure prediction, chemical protection and PKR activation assays. Methods for the selection of active PKR RNA complexes from mixtures of active and inactive complexes are proposed. The inherent binding selectivity for the 20 kDa RNA-binding domain (RBD) will be defined by identification of ligands through selections from RNA libraries. In addition, binding by the PKR RBD to synthetic RNA duplexes varying in sequence and additional secondary structural elements, such as signal base bulges and hairpin loops, will be characterized by RNA affinity cleaving with EDTA Fe modified proteins.
