IDENTIFICATION AND REQUIREMENT OF 3 RIBOSOME BINDING DOMAINS IN DSRNA-DEPENDENT PROTEIN-KINASE (PKR)

The interferon-inducible, double-stranded (ds) RNA-dependent protein k inase (PKR) regulates protein synthesis initiation by phosphorylating the alpha-subunit of eukaryotic translation initiation factor 2 (eIF-2 ). The amino-terminal half of PKR contains two dsRNA binding domains, and the kinase domain resides in the carboxy-terminal half of the prot ein. PKR is a ribosomal-associated protein. In this report, we provide evidence that PKR contains three ribosome interaction sites, two that are localized in each of the dsRNA binding domains and one that is lo calized in the kinase domain, All three domains can associate with pol ysomes independently. The ribosome association of the dsRNA binding do mains requires dsRNA binding activity. Ribosome interaction of either the individual or the combined dsRNA binding domains was disrupted by 0.1 M KCl. In contrast, the ribosome interaction of intact PKR and the isolated kinase domain was largely resistant to 0.5 M KCl. These resu lts ndicate that all three domains of PKR contribute to the high-affin ity ribosomal association. After dissociation of polysomes with EDTA, both intact PKR and the isolated kinase domain were primarily associat ed with the 60S ribosomal subunit. Coexpression of the adenovirus VAI RNA, an RNA polymerase III gene product that binds and inactivates PKR , disrupted ribosomal association of intact PKR, but not of the isolat ed PKR kinase domain. The results support a model where VAI RNA induce s a major conformational change in PKR to prohibit ribosome associatio n of all interaction sites. In contrast, other inhibitors of PKR inclu ding vaccinia virus E3L and K3L gene products, and the HIV trans-activ ating response (TAR) element binding protein TRBP, did not disrupt rib osome association of PKR. The results suggest a novel mechanism by whi ch viral RNAs may inactivate PKR through disrupting ribosome associati on.