eEF2 diphthamide modification restrains spurious frameshifting to maintain translational fidelity.

Diphthamide (DPH), a conserved amino acid modification on eukaryotic translation elongation factor eEF2, is synthesized via a complex, multi-enzyme pathway. While DPH is non-essential for cell viability and its function has not been resolved, diphtheria and other bacterial toxins ADP-ribosylate DPH to inhibit translation. Characterizing Saccharomyces cerevisiae mutants that lack DPH or show synthetic growth defects in the absence of DPH, we show that loss of DPH increases resistance to the fungal translation inhibitor sordarin and increases -1 ribosomal frameshifting at non-programmed sites during normal translation elongation and at viral programmed frameshifting sites. Ribosome profiling of yeast and mammalian cells lacking DPH reveals increased ribosomal drop-off during elongation, and removal of out-of-frame stop codons restores ribosomal processivity on the ultralong yeast MDN1 mRNA. Finally, we show that ADP-ribosylation of DPH impairs the productive binding of eEF2 to elongating ribosomes. Our results reveal that loss of DPH impairs the fidelity of translocation during translation elongation resulting in increased rates of ribosomal frameshifting throughout elongation and leading to premature termination at out-of-frame stop codons. We propose that the costly, yet non-essential, DPH modification has been conserved through evolution to maintain translational fidelity despite being a target for inactivation by bacterial toxins.