PKR is activated by and binds tightly to A14G mt-tRNA^Leu dimers.

<p>(A) Activation assays for native gel purified WT monomer and A14G dimer of mt-tRNA^Leu. RNA concentrations are 0.04, 0.08, 0.16, 0.32, 0.63, 1.25, 2.5, and 5 µM. A no RNA control and a positive control of 0.1 µM dsRNA-79 are included. Phosphorylation activities are provided below each gel lane. Phosphorylation activities were normalized to dsRNA-79 and rounded to the nearest integer. (B, C) Binding assays for dsRBD of PKR (P20) and WT and A14G mt-tRNA^Leu. Trace amount of 5′-³²P-labeled tRNA was used in the binding experiments. Protein concentrations are 0, 0.04, 0.08, 0.16, 0.32, 0.63, 1.25, 2.5, 5, 10 and 20 µM and are present in Lanes 1 to 11, respectively. For one set of binding experiments (B) herring sperm DNA competitor was used (0.1 mg/mL) and for the other (C) tRNA competitor was used (0.1 mg/mL). Binding constants for A14G (○) and WT (•) in DNA competitor were 1.3 and 5.1 µM with Hill coefficients of 3.7 and 2.9 respectively, and in the presence of tRNA competitor they were 3.1 and >20 µM with Hill coefficients of 3.1 and undetermined, respectively. Higher Hill coefficients correlate with multiple bands of lower mobility on native gels, especially in Panel C (14G); this may relate to the dimer having enough binding registers to accommodate multiple copies of P20 at one time.