SUMMARY Oxidative stress causes K63-linked ubiquitination of ribosomes by the E2 ubiquitin conjugase, Rad6. How Rad6-mediated ubiquitination of ribosomes affects global translation, however, is unclear. We therefore performed Ribo-seq and Disome-seq in Saccharomyces cerevisiae, and found that oxidative stress caused ribosome pausing at specific amino acid motifs, and this also led to ribosome collisions. However, these redox pausing signatures were lost in the absence of Rad6 but did not depend on the ribosome-associated quality control (RQC) pathway. We also found that Rad6 is needed to inhibit overall translation in response to oxidative stress and its deletion leads to increased expression of antioxidant genes. Finally, we observed that the lack of Rad6 leads to changes during translation initiation that affect activation of the integrated stress response (ISR) pathway. Our results provide a high-resolution picture of the gene expression changes during oxidative stress and unravel an additional stress response pathway affecting translation elongation. HIGHLIGHTS Rad6 is required for sequence-specific ribosome pausing under oxidative stress. Rad6 affects translation independently of the RQC pathway. Cells lacking Rad6 show dysregulated translational repression upon oxidative stress. Loss of Rad6 leads to altered activation of the ISR pathway.

The ubiquitin conjugase Rad6 mediates ribosome pausing during oxidative stress