Mechanistic characterization of the 5′-triphosphate-dependent activation of PKR: Lack of 5′-end nucleobase specificity, evidence for a distinct triphosphate binding site, and a critical role for the dsRBD

Photochemical cross-linking reveals interaction of ppp-ssRNA-47 with PKR regions outside the dsRBD. 4thioU-substituted ppp-ssRNA-47 was incubated with wild-type PKR, P20, or dmPKR and exposed to 365-nm light for 0, 10, 20, or 30 min and analyzed by 7% denaturing (7 M urea) PAGE. The positions of free RNA and cross-linked products are indicated. Protein dependence of cross-linked products was confirmed by the absence of products upon irradiating 4-thioU-ppp-ssRNA-47 in buffer alone (first three lanes).