Characterization of the solution complex between the interferon-induced, double-stranded RNA-activated protein kinase and HIV-I trans-activating region RNA.
The antiviral activity of the interferon-induced, double-stranded RNA (dsRNA)-activated protein kinase (PKR) is mediated through dsRNA binding leading to PKR autophosphorylation and subsequent inhibition of protein synthesis. Previous biochemical studies have suggested that autophosphorylation of PKR occurs via a protein-protein interaction and that PKR can form dimers in vitro. Using four independent biophysical and biochemical methods, we have characterized the solution complex formed between PKR and trans-activating region (TAR) RNA, a 57-nucleotide RNA species with double-stranded secondary structure derived from the human immunodeficiency virus type I genome. Chemical cross-linking and gel filtration analyses of PKR.TAR RNA complexes reveals that TAR RNA addition increases PKR dimerization and results in the formation of a solution complex with a molecular weight of approximately 150,000. Addition of TAR RNA to PKR results in a quenching of tryptophan fluorescence, indicative of a conformational shift. Through small angle neutron scattering analysis, we show that PKR exists in solution predominantly as a dimer, and has an elongated solution structure. Addition of TAR RNA to PKR causes a significant conformational shift in the protein at a 2:1 stoichiometric ratio of protein to RNA. Taken together, these data indicate that the PKR activation complex consists of a protein dimer bound cooperatively to one dsRNA molecule.
Carpick
B W
BW
Department of Cancer Biology, Research Institute, The Cleveland Clinic Foundation, Cleveland, Ohio 44195, USA.
Graziano
V
V
Schneider
D
D
Maitra
R K
RK
Lee
X
X
Williams
B R
BR
D001483:Base Sequence; D002850:Chromatography, Gel; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004120:Dimethyl Suberimidate / Q000494:pharmacology; D016325:HIV Long Terminal Repeat; D015497:HIV-1; D006801:Humans; D008969:Molecular Sequence Data; D009502:Neutrons; D009690:Nucleic Acid Conformation; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000378:metabolism*; D012330:RNA, Double-Stranded / Q000737:chemistry / Q000378:metabolism*; D012367:RNA, Viral / Q000737:chemistry / Q000378:metabolism*; D012542:Scattering, Radiation; D013050:Spectrometry, Fluorescence; D019892:eIF-2 Kinase
D003432:Cross-Linking Reagents; D012330:RNA, Double-Stranded; D012367:RNA, Viral; D004120:Dimethyl Suberimidate; D017346:Protein Serine-Threonine Kinases; D019892:eIF-2 Kinase