Fig. 3: PKR scans along dsRNA, resulting in dsRNA length-dependent activity.
From: PACT prevents aberrant activation of PKR by endogenous dsRNA without sequestration
A In vitro PKR kinase assay monitoring PKR autophosphorylation with increasing concentrations (0.125, 0.25, 2.5 ng/μl) of 112 bp dsRNA with 4 different sequences (see supplementary Table 3) and GC contents. [35S]-IRF3 served as loading control (*). Wild-type full-length PKR was used unless stated otherwise. Results were reproduced in 2 replicates. B PKR kinase assay with increasing dsRNA concentrations (0.0625–15 ng/μl) for lengths 112, 62, 42, and 21 bp. Results reproduced in 3 replicates. C, D Quantification of PKR activity from A displayed as heatmap and x-y graph. Data represent mean ( ± SD) of 3 biological repeats. E PKR activity with 112 bp dsRNA, perfect duplex Alu dsRNA, and IR-Alus from NICN1 and PSMB2 3’-UTRs (see Supplementary Fig. 5D) at 0.0625, 0.25, and 1 ng/μl. Values are mean ( ± SD) of 3 biological replicates. F Schematic of single-molecule FRET experiments to monitor scanning motions of PKR on dsRNA. Cy5-labeled PKR (acceptor, 50 nM) was added to a chamber containing Cy3-labeled dsRNA (donor) immobilized on the surface. Cy3 and Cy5 fluorescence were monitored upon Cy3 excitation using sm-TIRF microscopy. Time course traces of Cy3/Cy5 intensities and FRET (right panel) and zoomed-in trace for “On” state (lower panel). PKR K296R was used to ensure analysis of PKR movement before its activation. *indicates transient or abortive binding––such events were excluded from the autocorrelation analysis in G. See Supplementary Fig. 6 for additional traces and description of identifying “On” events. Created in BioRender. torres, c. (2025) https://BioRender.com/g96x368. G Autocorrelation analysis of FRET fluctuations for PKR in the “On” state on 112, 59, 42 bp dsRNAs and “Off” state on 112 bp dsRNA. See Methods for the autocorrelation function. (H) Diffusion rates (mean ± SD) calculated from exponential fitting of the autocorrelation curves of PKR (see Methods) with different dsRNA lengths: 0.130 ± 0.00657 s (42 bp), 0.149 ± 0.00657 s (59 bp) and 0.230 ± 0.0101 s (112 bp); n = 143 (42 bp), 69 (59 bp) and 236 (112 bp) diffusion events. Source data is provided as a Source Data file.
