RNA binding and modulation of PKR activity.
PKR is an RNA-dependent protein kinase that is induced in mammalian cells by interferon treatment. It is present in a latent or inactive form in mammalian cells and is activated by very low concentrations of double-stranded (ds) RNA. Activated PKR phosphorylates eIF2, an essential initiation factor of protein synthesis, as well as other substrates including histone IIA, a 90-kDa protein from rabbit reticulocytes, the inhibitor, IkappaB, of the transcription factor, NF-kappaB, and the HIV-1 Tat protein. PKR interacts with several cellular and viral products 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.
Gunnery
S
S
Department of Biochemistry and Molecular Biology, New Jersey Medical School, UMDNJ, 185 South Orange Avenue, University Heights, Newark, New Jersey, 07103, USA.
Mathews
M B
MB
D002853:Chromatography, Liquid / Q000379:methods; D004789:Enzyme Activation; D015852:Eukaryotic Initiation Factor-2 / Q000378:metabolism; D015696:Gene Products, tat / Q000378:metabolism; D015497:HIV-1 / Q000378:metabolism; D006657:Histones / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism; D010766:Phosphorylation; D012313:RNA / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism*; D019892:eIF-2 Kinase / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D054322:tat Gene Products, Human Immunodeficiency Virus
D015852:Eukaryotic Initiation Factor-2; D015696:Gene Products, tat; D006657:Histones; D016328:NF-kappa B; D016601:RNA-Binding Proteins; D054322:tat Gene Products, Human Immunodeficiency Virus; D012313:RNA; D019892:eIF-2 Kinase