Translation is highly regulated. Phosphorylation of translation initiation factors has been shown to be a key feature of regulation in eukaryotes. Double-stranded RNA dependent protein kinase (PKR) is a 68 kDa protein that is activated by dsRNA binding, leading to phosphorylation of initiation factor elF2a. Phosphorylation of elF2 leads to blockage of nucleotide exchange by elF2B, resulting in down regulation of translation. PKR function has been linked to innate immune antiviral response, and its activity is modulated in many cancers. Viral RNAs can activate PKR, and several viruses encode RNAs and proteins that inhibit PKR function. Despite a decade of biochemical and structural studies, the basis for RNA activation of PKR is not known. Here we propose to study the linkage between double-stranded RNA binding and kinase activation.
In specific aim 1, we will define a set of RNA activators and inhibitors of PKR that bind specifically to the tandem double-stranded RNA binding motifs (dsRBD1,2) of PKR. We will determine the thermodynamic parameters that govern RNA-protein recognition, and establish defined systems for structural studies.
In specific aim 2, the structures of dsRBD1,2 in complex with several of these viral RNAs-HlV TAR, Epstein Barr virus EBER and adenovirus VAI, will be determined by NMR to establish the structural basis for RNA-protein recognition.
In specific aim 3, the mechanism of PKR will be investigated using a variety of biochemical and biophysical methods. We will delineate the kinetic framework for kinase function, and compare the action of RNA activators and inhibitors.
In specific aim 4, we will study the structural features and conformational dynamics of full-length PKR using NMR spectroscopy and single-molecule fluorescence approaches. We will attempt to link the kinetic observations of aim 3 with changes in PKR conformation and aggregation state as a function of RNA binding and autophosphorylation. Finally in specific aim 5, we will link the molecular mechanism for RNA activation of PKR with the effects of PKR on translation in extracts. The proposal links basic physical principles of RNA-protein recognition with a molecular view of PKR activation and effects on translation. The results of this work will explain a central mechanism of gene regulation required for antiviral response and control of cell proliferation. ? ? ?
|Elazar, Menashe; Liu, Michael; McKenna, Sean A et al. (2009) The anti-hepatitis C agent nitazoxanide induces phosphorylation of eukaryotic initiation factor 2alpha via protein kinase activated by double-stranded RNA activation. Gastroenterology 137:1827-35|
|Shimoike, Takashi; McKenna, Sean A; Lindhout, Darrin A et al. (2009) Translational insensitivity to potent activation of PKR by HCV IRES RNA. Antiviral Res 83:228-37|
|McKenna, Sean A; Lindhout, Darrin A; Shimoike, Takashi et al. (2007) Viral dsRNA inhibitors prevent self-association and autophosphorylation of PKR. J Mol Biol 372:103-13|
|McKenna, Sean A; Lindhout, Darrin A; Kim, Insil et al. (2007) Molecular framework for the activation of RNA-dependent protein kinase. J Biol Chem 282:11474-86|