Mechanism of interferon action motif I of the interferon-induced, RNA-dependent protein kinase (PKR) is sufficient to mediate RNA-binding activity.
The interferon-induced P1/eIF-2 alpha protein kinase cDNA, designated PKR, was expressed both in Escherichia coli and in transfected monkey COS cells. TrpE-PKR fusion proteins and PKR nonfusion proteins were examined for their RNA-binding activity by Northwestern blot analysis. PKR is a RNA-binding protein that possesses two copies of a highly conserved motif, RI and RII, within the N-terminal region of the protein. Amino acid residues between 11 and 243 of PKR, which includes both copies of the R motif, displayed RNA-binding activity comparable to that of the full-length 551-amino-acid PKR protein. Analysis of substitution and deletion mutant PKR proteins revealed that motif RI was both necessary and sufficient for RNA-binding activity, whereas motif RII was not. Substitution of the highly conserved lysine at position 64 within the RI motif abolished RNA-binding activity, both of full-length PKR and the N-terminal 243-amino-acid truncated PKR. Finally, PKR substitution and deletion mutant cDNAs deficient for kinase function were expressed to much higher levels in transfected monkey cells than was the full-length wild-type PKR cDNA.
McCormack
S J
SJ
Department of Biological Sciences, University of California at Santa Barbara 93106.
Ortega
L G
LG
Doohan
J P
JP
Samuel
C E
CE
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002522:Chlorocebus aethiops; D004790:Enzyme Induction / Q000187:drug effects; D015870:Gene Expression; D007372:Interferons / Q000494:pharmacology*; D008969:Molecular Sequence Data; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014162:Transfection; D019892:eIF-2 Kinase
D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D007372:Interferons; D017346:Protein Serine-Threonine Kinases; D019892:eIF-2 Kinase