Abstract
Eukaryotic translation elongation factor 2 (eEF2) facilitates the movement of the peptidyl tRNA-mRNA complex from the A site of the ribosome to the P site during protein synthesis. ADP-ribosylation (ADP(R)) of eEF2 by bacterial toxins on a unique diphthamide residue inhibits its translocation activity, but the mechanism is unclear. We have employed a hormone-inducible diphtheria toxin (DT) expression system in Saccharomyces cerevisiae which allows for the rapid induction of ADP(R)-eEF2 to examine the effects of DT in vivo. ADP(R) of eEF2 resulted in a decrease in total protein synthesis consistent with a defect in translation elongation. Association of eEF2 with polyribosomes, however, was unchanged upon expression of DT. Upon prolonged exposure to DT, cells with an abnormal morphology and increased DNA content accumulated. This observation was specific to DT expression and was not observed when translation elongation was inhibited by other methods. Examination of these cells by electron microscopy indicated a defect in cell separation following mitosis. These results suggest that expression of proteins late in the cell cycle is particularly sensitive to inhibition by ADP(R)-eEF2.
Highlights
Diphtheria toxin inhibits translation by ADP-ribosylation of elongation factor 2
The diphthamide residue resulting from post-translational modification of a conserved histidine in eukaryotic EF2 is the known site of action of diphtheria toxin (DT)
This dominant resistance to toxin suggests that DTR-Eukaryotic translation elongation factor 2 (eEF2) is functional despite the presence of nonfunctional ADPR-eEF2; the expression and ADP-ribosylation of wild-type eEF2 was not confirmed under these conditions
Summary
Diphtheria toxin inhibits translation by ADP-ribosylation of elongation factor 2. Upon prolonged exposure to DT, cells with an abnormal morphology and increased DNA content accumulated This observation was specific to DT expression and was not observed when translation elongation was inhibited by other methods. Cryo-EM structures of eEF2 bound to the ribosome demonstrated that the tip of domain IV, which constitutes the anticodon mimic site of eEF2, is located near the ribosomal A site and plays an important role in translocation and translational fidelity [3, 4] Positioned within this region of domain IV is a unique diphthamide residue that results from the multistep enzymatic conversion of a single histidine (His-715 and His-699 in humans and yeast, respectively) [5,6,7].
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