Identification of the double-stranded RNA-binding domain of the human interferon-inducible protein kinase.
The interferon-inducible double-stranded (ds) RNA-activated protein kinase (p68 kinase) is a physiologically important enzyme that regulates the rate of cellular and viral protein synthesis by phosphorylating and thereby inactivating the peptide chain initiation factor 2. We have generated a cDNA clone of the human p68 kinase by polymerase chain reaction cloning using the recently published sequence of this enzyme. Active enzyme was synthesized by in vitro transcription-translation of the cDNA clone. This system was used for mapping the dsRNA-binding domain of the enzyme. Progressive deletions from the carboxyl terminus were introduced by digesting the cDNA with suitable restriction enzymes. Expression of proteins harboring deletions from the amino terminus was achieved by cloning DNA fragments into appropriately constructed expression vectors. Affinity of the truncated proteins for dsRNA was examined by testing their capacity to bind to dsRNA-agarose beads. Our results demonstrated that the dsRNA-binding domain lies at the amino terminus of the protein. A truncated protein containing the first 170 amino acid residues from the amino terminus could bind to dsRNA. However, deletion of 34 residues from the amino terminus or 41 residues from the carboxyl terminus of this truncated protein eliminated its dsRNA-binding activity. Comparison of the primary structure and the secondary structure of this region of p68 kinase and the corresponding region of 2'-5'-oligoadenylate synthetase revealed no apparent similarity.
Department of Molecular Biology, Cleveland Clinic Foundation, Ohio 44195.
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D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D004247:DNA; D004587:Electrophoresis, Agar Gel; D004790:Enzyme Induction; D005822:Genetic Vectors; D006801:Humans; D007372:Interferons / Q000494:pharmacology; D008969:Molecular Sequence Data; D010766:Phosphorylation; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D011494:Protein Kinases / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D012330:RNA, Double-Stranded / Q000378:metabolism*; D014158:Transcription, Genetic; D019892:eIF-2 Kinase
D012330:RNA, Double-Stranded; D004247:DNA; D007372:Interferons; D011494:Protein Kinases; D019892:eIF-2 Kinase