RNA-BINDING AND MODULATION OF PKR ACTIVITY

PKR is an RNA-dependent protein kinase that is induced in mammalian ce lls by interferon treatment. It is present in a latent or inactive for m in mammalian cells and is activated by very low concentrations of do uble-stranded (ds) RNA. Activated PKR phosphorylates elF2, an essentia l initiation factor of protein synthesis, as well as other substrates including histone IIA, a 90-kDa protein from rabbit reticulocytes, the inhibitor, I kappa B, of the transcription factor, NF-kappa B, and th e HIV-1 Tat protein. PKR interacts with several cellular and viral pro ducts and these interactions modulate its activation by dsRNA. Here we describe methods that are used to study the activation or inhibition of PKR by RNA modulators. Specifically, we detail (1) the purification of PKR from interferon-treated mammalian cells, (2) functional assays for PKR activation and inhibition in vitro, using purified enzyme or crude cell lysates, and (3) assays allowing evaluation of the binding of dsRNA and single-stranded RNA to PKR. (C) 1998 Academic Press.