Specific mutations near the amino terminus of double-stranded RNA-dependent protein kinase (PKR) differentially affect its double-stranded RNA binding and dimerization properties.
The amino-terminal region of the double-stranded (ds) RNA-dependent protein kinase, PKR, has been shown to mediate both dsRNA binding and protein dimerization. To critically examine if PKR dimerization is dependent on dsRNA binding, we generated a series of mutants that are incapable of binding dsRNA. Some, but not all, of these mutants retained the ability to dimerize, as shown by a two-hybrid transcriptional activation assay in vivo and a chemical cross-linking assay in vitro. These mutants were used further to demonstrate that the translational inhibitory activity of PKR in vivo requires dsRNA binding; PKR mutants that dimerized but did not bind dsRNA could not inhibit the translation of a transfected reporter gene.
Patel
R C
RC
Department of Molecular Biology, Cleveland Clinic Foundation, Cleveland, Ohio 44195, USA.
Stanton
P
P
Sen
G C
GC
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D019556:COS Cells; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D019281:Dimerization; D017930:Genes, Reporter; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry* / Q000378:metabolism*; D012330:RNA, Double-Stranded / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D017384:Sequence Deletion; D015533:Transcriptional Activation; D014162:Transfection; D019892:eIF-2 Kinase
D009838:Oligodeoxyribonucleotides; D012330:RNA, Double-Stranded; D011993:Recombinant Fusion Proteins; D017346:Protein Serine-Threonine Kinases; D019892:eIF-2 Kinase