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 protein s that interact with structured. RNAs. We have expressed the entire do main and the first copy of the motif in Escherichia coli and purified the two proteins, p20 and p10, to apparent homogeneity in order to stu dy their interactions with RNA and with the intact kinase enzyme. Both p20 and p10 bound preferentially to structured RNA molecules. Competi tion assays showed that in both cases the order of affinity is dsRNA > VA RNA > tRNA, but the isolated motif bound much less tightly than th e entire domain. Measurement of the dissociation constants for dsRNA b y quantitative gel mobility shift analysis gave apparent K-d values of 4 x 10(-9) M and 3.8 x 10(-7) M for p20 and p10, respectively. The bi nding of p20 molecules to dsRNA appeared to be cooperative. Multiple c omplexes were formed between the intact domain and dsRNA, saturating a t a density of about one p20 molecule/11.25 base-pairs (or one turn) o f duplex, whereas p10 achieved only about half of this packing density : The apparent K-d for the p20-VA RNA interaction was estimated as 3.5 x 10(-7) M and at least three complexes were detected, but no distinc t complexes were visualized for the interaction between p10 and VA RNA . Both p20 and p10 inhibited autophosphorylation of intact DAI, probab ly by binding the dsRNA activator. Once activated, DAI could phosphory late both p10 and p20, suggesting that intermolecular phosphorylation can occur.