Functional characterization of the RNA-binding domain and motif of the double-stranded RNA-dependent protein kinase DAI (PKR).
The double-stranded (ds) RNA-activated protein kinase, DAI (also known as PKR), contains an RNA-binding domain comprising two tandem repeats of a motif, the dsRBM, which is shared with a number of other proteins that interact with structured RNAs. We have expressed the entire domain and the first copy of the motif in Escherichia coli and purified the two proteins, p20 and p10, to apparent homogeneity in order to study their interactions with RNA and with the intact kinase enzyme. Both p20 and p10 bound preferentially to structured RNA molecules. Competition assays showed that in both cases the order of affinity is dsRNA > VA RNA > tRNA, but the isolated motif bound much less tightly than the entire domain. Measurement of the dissociation constants for dsRNA by quantitative gel mobility shift analysis gave apparent Kd values of 4 x 10(-9) M and 3.8 x 10(-7) M for p20 and p10, respectively. The binding of p20 molecules to dsRNA appeared to be cooperative. Multiple complexes were formed between the intact domain and dsRNA, saturating at a density of about one p20 molecule/11.25 base-pairs (or one turn) of duplex, whereas p10 achieved only about half of this packing density. The apparent Kd for the p20-VA RNA interaction was estimated as 3.5 x 10(-7) M and at least three complexes were detected, but no distinct complexes were visualized for the interaction between p10 and VA RNA. Both p20 and p10 inhibited autophosphorylation of intact DAI, probably by binding the dsRNA activator. Once activated, DAI could phosphorylate both p10 and p20, suggesting that intermolecular phosphorylation can occur.
Schmedt
C
C
Cold Spring Harbor Laboratory, NY 11724, USA.
Green
S R
SR
Manche
L
L
Taylor
D R
DR
Ma
Y
Y
Mathews
M B
MB
D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D008969:Molecular Sequence Data; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D012330:RNA, Double-Stranded / Q000378:metabolism*; D019892:eIF-2 Kinase
D012330:RNA, Double-Stranded; D017346:Protein Serine-Threonine Kinases; D019892:eIF-2 Kinase