Eukaryotic elongation factor 2 (eEF2) mediates translocation in protein synthesis. The molecular mimicry model proposes that the tip of domain IV mimics the anticodon loop of tRNA. His-699 in this region is post-translationally modified to diphthamide, the target for Corynebacterium diphtheriae and Pseudomonas aeruginosa toxins. ADP-ribosylation by these toxins inhibits eEF2 function causing cell death. Mutagenesis of the tip of domain IV was used to assess both functions. A H694A mutant strain was non-functional, whereas D696A, I698A, and H699N strains conferred conditional growth defects, sensitivity to translation inhibitors, and decreased total translation in vivo. These mutant strains and those lacking diphthamide modification enzymes showed increased ؊1 frameshifting. The effects are not due to reduced protein levels, ribosome binding, or GTP hydrolysis. Functional eEF2 forms substituted in domain IV confer dominant diphtheria toxin resistance, which correlates with an in vivo effect on translation-linked phenotypes. These results provide a new mechanism in which the translational machinery maintains the accurate production of proteins, establishes a role for the diphthamide modification, and provides evidence of the ability to suppress the lethal effect of a toxin targeted to eEF2.The eukaryotic translation elongation factor 2 (eEF2) 2 and its bacterial homolog elongation factor G (EF-G) are members of the G-protein superfamily. These two proteins catalyze the translocation step of translation elongation after peptide bond formation occurs. The tRNAs located in the A-and P-sites are translocated to the P-and E-sites followed by the advancement of three bases of the mRNA to allow another round of translation elongation (reviewed in Ref. 1). In the yeast Saccharomyces cerevisiae, eEF2 is encoded by two genes, EFT1 and EFT2. The encoded proteins are identical, and one must be present for viability (2).Even though work on EF-G has proven to be invaluable in our understanding of the function of eEF2 on protein synthesis, marked differences are evident between the two homologous proteins. The most pronounced are the post-translation modifications that occur on eEF2. These modifications are the phosphorylation of Thr-57 and the diphthamide modification of His-699 in yeast and His-715 in mammals. S. cerevisiae eEF2 is phosphorylated by the Rck2p kinase (3), a Ser/Thr protein kinase homologous to the mammalian calmodulin kinases, which requires phosphorylation for activation (4, 5). In mammalian cells, eEF2 is phosphorylated on Thr-57 by the eEF2 kinase, a Ca 2ϩ /calmodulin-dependent protein kinase (6). The unique diphthamide modification is the result of a multistep conversion requiring several enzymatic activities performed by the DPH gene products in yeast (7). This modification is located at the tip of domain IV of the protein (8), a region proposed to mimic the tRNA anticodon loop (reviewed in Ref. 9). Although phosphorylation reduces the affinity for GTP, but not GDP, and decreases ribosome binding (10), a r...