REQUIREMENT OF PKR DIMERIZATION MEDIATED BY SPECIFIC HYDROPHOBIC RESIDUES FOR ITS ACTIVATION BY DOUBLE-STRANDED-RNA AND ITS ANTIGROWTH EFFECTS IN YEAST

The roles of protein dimerization and double-stranded RNA (dsRNA) bind ing in the biochemical and cellular activities of PKR, the dsRNA-depen dent protein kinase, were investigated. We have previously shown that both properties of the protein are mediated by the same domain. Here w e show that dimerization is mediated by hydrophobic residues present o n one side of an amphipathic oc-helical structure within this domain. Appropriate substitution mutations of residues on that side produced m utants with increased or decreased dimerization activities. Using thes e mutants, we demonstrated that dimerization is not essential for dsRN A binding. However, enhancing dimerization artificially, by providing an extraneous dimerization domain, increased dsRNA binding of both wil d-type and mutant proteins. In vitro, the dimerization-defective mutan ts could not be activated by dsRNA but were activated normally by hepa rin. In Saccharomyces cerevisiae, unlike wild-type PKR, these mutants could not inhibit cell growth and the dsRNA-binding domain of the dime rization-defective mutants could not prevent the antigrowth effect of wild-type PKR. These results demonstrate the biological importance of the dimerization properties of PKR.