Mechanism of RACK1-dependent ZAKɑ activation at stalled and collided ribosomes

Combining Alphafold3 prediction and RNA crosslinking and immunoprecipitation (CLIP), we uncover that ZAKɑ dynamically monitors the mRNA exit channel of elongating ribosomes. This is accomplished by ZAKɑ via its direct interactions with the ribosomal proteins RACK1 and RPS27 as well as with the 18S rRNA helix-26.

In this conformation, four RNA-binding peptides in ZAKɑ span across the path of ribosome-exiting mRNA. Progressive elongation effectively threads ZAKɑ off the ribosome, while mRNA stasis stabilizes the interaction allowing for kinase activation. Prolonged binding of ZAKɑ to slow-elongating, stalled and collided ribosomes is associated with sequestration of the SAM domain on RACK1, allowing for transient ZAKɑ dimerization, activation loop trans-autophosphorylation and RSR activation.