Characterization of the interactions between double-stranded RNA and the double-stranded RNA binding domain of the interferon induced protein kinase.
The interferon-inducible protein kinase, PKR, requires double-stranded (ds) RNA for its activation. We have previously mapped its dsRNA-binding domain (DRBD) to the amino terminal 170 residues (Patel and Sen, 1992). In the present study, we have characterized in detail the interactions between dsRNA and DRBD. For this purpose, DRBD was produced in bacteria as a polyhistidine-tagged protein and purified by affinity chromatography. A polyclonal antibody was raised against purified DRBD. For studying dsRNA-DRBD interactions, a Northwestern assay and an electrophoretic mobility shift assay (EMSA) using a radiolabeled in vitro transcribed 82 bp dsRNA probe was developed. The antiserum reacted with both DRBD and PKR but did not prevent their interactions with dsRNA. DRBD, on the other hand, blocked the activation of PKR by dsRNA. DRBD and the dsRNA probe formed multimeric complexes which were separable by EMSA. The antibody could interact with these complexes and supershift their mobility. Competition with unlabeled dsRNA revealed that the dimeric DRBD-dsRNA complex was much more stable than the monomeric complex. Similar competition assays using 11 different synthetic and natural RNA molecules revealed that only authentic dsRNA molecules could effectively compete with the probe for binding DRBD in a sequence-independent fashion.
Patel
R C
RC
Department of Molecular Biology, Cleveland Clinic Foundation, OH 44195-5285, USA.
Sen
G C
GC
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D006639:Histidine; D007106:Immune Sera; D008969:Molecular Sequence Data; D010455:Peptides; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D012330:RNA, Double-Stranded / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D016324:Sequence Tagged Sites; D014158:Transcription, Genetic; D019892:eIF-2 Kinase
D017931:DNA Primers; D007106:Immune Sera; D010455:Peptides; D012330:RNA, Double-Stranded; D011994:Recombinant Proteins; C033223:polyhistidine; D006639:Histidine; D017346:Protein Serine-Threonine Kinases; D019892:eIF-2 Kinase