Translation Elongation Factor 2 Anticodon Mimicry Domain Mutants Affect Fidelity and Diphtheria Toxin Resistance

Abstract

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...

Translation Elongation Factor 1A Mutants with Altered Actin Bundling Activity Show Reduced Aminoacyl-tRNA Binding and Alter Initiation via eIF2α Phosphorylation

“…Therefore, we wanted to determine whether a general defect in translation would be enough to cause an increase in eIF2␣ phosphorylation levels. Analysis of other eEF1A as well as eEF2 and eEF3 mutants known to affect translation elongation (29,30) showed that increased eIF2␣ phosphorylation does not result from a general reduction in translation elongation (Fig. 1B).…”

“…1B). To determine whether the phosphorylation effect was due to a general inhibition of translation, we analyzed the level of eIF2␣ phosphorylation in eEF2 and eEF3 mutants with established translation elongation defects (29,30). Neither of the eEF2 and eEF3 mutants gave rise to increased eIF2␣ phosphorylation (Fig.…”

Diphthamide modification on eukaryotic elongation factor 2 is needed to assure fidelity of mRNA translation and mouse development

“…A recent study on yeast eEF2 mutants provided evidence that diphthamide plays a role in maintaining the fidelity in protein translation on ribosomes (29). The yeast mutant strains lacking diphthamide modification enzymes show modestly increased −1 frameshifting, an effect not due to a reduction in protein synthesis, ribosome binding, or GTP hydrolysis (29). Remarkably, here we found not only OVCA1 −/− MEFs but also eEF2 G717R/G717R MEFs showed significant increases in −1 frameshifting during translation compared with WT cells.…”

Mechanism and Regulation of Protein Synthesis in Saccharomyces cerevisiae

“…Much analysis has focused on His699, the modified residue (see below) at the tip. Viable mutants that alter His699 or other residues within the tip of domain IV cause modest defects in translational fidelity, including increased programmed 21 frameshifting (Ortiz et al 2006). Analysis of the site of binding of Sordarin, a natural product inhibitor of eEF2, via mutational and structural analyses, has demonstrated that the compound binds between domains I, III, and V of eEF2, matching the sites of Sordarin-resistant mutations (Justice et al 1998;Soe et al 2007).…”