Two crystal structures of yeast translation elongation factor 2 (eEF2) were determined: the apo form at 2.9 A resolution and eEF2 in the presence of the translocation inhibitor sordarin at 2.1 A resolution. The overall conformation of apo eEF2 is similar to that of its prokaryotic homolog elongation factor G (EF-G) in complex with GDP. Upon sordarin binding, the three tRNA-mimicking C-terminal domains undergo substantial conformational changes, while the three N-terminal domains containing the nucleotide-binding site form an almost rigid unit. The conformation of eEF2 in complex with sordarin is entirely different from known conformations observed in crystal structures of EF-G or from cryo-EM studies of EF-G-70S complexes. The domain rearrangements induced by sordarin binding and the highly ordered drug-binding site observed in the eEF2-sordarin structure provide a high-resolution structural basis for the mechanism of sordarin inhibition. The two structures also emphasize the dynamic nature of the ribosomal translocase.

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...

The crystal structure of the N-terminal 219 residues (domain 1) of the conserved eukaryotic translation elongation factor 1B␥ (eEF1B␥), encoded by the TEF3 gene in Saccharomyces cerevisiae, has been determined at 3.0 Å resolution by the single wavelength anomalous dispersion technique. The structure is overall very similar to the glutathione S-transferase proteins and contains a pocket with architecture highly homologous to what is observed in glutathione S-transferase enzymes. The TEF3-encoded form of eEF1B␥ has no obvious catalytic residue. However, the second form of eEF1B␥ encoded by the TEF4 gene contains serine 11, which may act catalytically. Based on the x-ray structure and gel filtration studies, we suggest that the yeast eEF1 complex is organized as an [eEF1A⅐eEF1B␣⅐eEF1B␥] 2 complex. A 23-residue sequence in the middle of eEF1B␥ is essential for the stable dimerization of eEF1B␥ and the quaternary structure of the eEF1 complex.

The multi-subunit guanine nucleotide exchange factor eEF1B for Saccharomyces cerevisiae Translation Elongation Factor 1A (eEF1A) has catalytic (eEF1Bα) and noncatalytic (eEF1Bγ) subunits. Deletion of the two nonessential genes encoding eEF1Bγ has no dramatic effects on total protein synthesis or translational fidelity. Instead, loss of each gene gives resistance to oxidative stress, and loss of both is additive. The level of stress resistance is similar to overexpression of the Yap1p stress transcription factor and is dependent on the presence of the YAP1gene. Cells lacking the catalytic eEF1Bα subunit show even greater resistance to CdSO 4 , with or without eEF1Bγ present. Thus, the loss of guanine nucleotide exchange activity promotes the resistance. As nucleotide exchange is a critical regulator of most G-proteins, these results indicate a new mechanism in the growing list of examples of post-transcriptional responses to cellular stress.

The eukaryotic translation elongation factor 2 (eEF2), a member of the G-protein superfamily, catalyzes the post-peptidyl transferase translocation of deacylated tRNA and peptidyl tRNA to the ribosomal E- and P-sites. eEF2 is modified by a unique post-translational modification: the conversion of His699 to diphthamide at the tip of domain IV, the region proposed to mimic the anticodon of tRNA. Structural models indicate a hinge is important for conformational changes in eEF2. Mutations of V488 in the hinge region and H699 in the tip of domain IV produce non-functional mutants that when co-expressed with the wild-type eEF2 result in a dominant-negative growth phenotype in the yeast Saccharomyces cerevisiae. This phenotype is linked to reduced levels of the wild-type protein, as total eEF2 levels are unchanged. Changes in the promoter, 5′-untranslated region (5′-UTR) or 3′-UTR of the EFT2 gene encoding eEF2 do not allow overexpression of the protein, showing that eEF2 levels are tightly regulated. The H699K mutant, however, also alters translation phenotypes. The observed regulation suggests that the cell needs an optimum amount of active eEF2 to grow properly. This provides information about a new mechanism by which translation is efficiently maintained.

Crystals of the Saccharomyces cerevisiae elongation factor 2 (eEF2) in complex with GDP were obtained with the vapour-diffusion technique after rapid puri®cation from industrial yeast. The crystals diffract to 2.85 A Ê and belong to the space group P2 1 2 1 2 1 . A yeast strain expressing a functional histidine-tagged eEF2 as the only form of the protein further allows facilitated puri®cation of the factor for both structural and functional studies.

Jet propulsion fuel-8 (JP-8) is a kerosene-based fuel that is used in military jets. The U.S. Armed Services and North Atlantic Treaty Organization countries adopted JP-8 as a standard fuel source and the U.S. military alone consumes more than 2.5 billion gallons annually. Preliminary epidemiologic data suggested that JP-8 may interact with noise to induce hearing loss, and animal studies revealed damage to presynaptic sensory cells in the cochlea. In the current study, Long-Evans rats were divided into four experimental groups: control, noise only, JP-8 only, and JP-8 + noise. A subototoxic level of JP-8 was used alone or in combination with a nondamaging level of noise. Functional and structural assays of the presynaptic sensory cells combined with neurophysiologic studies of the cochlear nerve revealed that peripheral auditory function was not affected by individual exposures and there was no effect when the exposures were combined. However, the central auditory nervous system exhibited impaired brainstem encoding of stimulus intensity. These findings may represent important and major shifts in the theoretical framework that governs current understanding of jet fuel and/or jet fuel + noise-induced ototoxicity. From an epidemiologic perspective, results indicate that jet fuel exposure may exert consequences on auditory function that may be more widespread and insidious than what was previously shown. It is possible that a large population of military personnel who are suffering from the effects of jet fuel exposure may be misidentified because they would exhibit normal hearing thresholds but harbor a "hidden" brainstem dysfunction.

Heavy metal tungsten alloys have replaced lead and depleted uranium in many munitions applications, due to public perception of these elements as environmentally unsafe. Tungsten materials left in the environment may become bioaccessible as tungstate, which might lead to population exposure through water and soil contamination. Although tungsten had been considered a relatively inert and toxicologically safe material, recent research findings have raised concerns about possible deleterious health effects after acute and chronic exposure to this metal. This investigation describes tissue distribution of tungsten in mice following oral exposure to sodium tungstate. Twenty-four 6-9 weeks-old C57BL/6 laboratory mice were exposed to different oral doses of sodium tungstate (0, 62.5, 125, and 200 mg/kg/d) for 28 days, and after one day, six organs were harvested for trace element analysis with inductively coupled plasma mass spectrometry (ICP-MS). Kidney, liver, colon, bone, brain, and spleen were analyzed by sector-field high-resolution ICP-MS. The results showed increasing tungsten levels in all organs with increased dose of exposure, with the highest concentration found in the bones and the lowest concentration found in brain tissue. Gender differences were noticed only in the spleen (higher concentration of tungsten in female animals), and increasing tungsten levels in this organ were correlated with increased iron levels, something that was not observed for any other organ or either of the two other metals analyzed (nickel and cobalt). These findings confirmed most of what has been published on tungsten tissue distribution; they also showed that the brain is relatively protected from oral exposure. Further studies are necessary to clarify the findings in splenic tissue, focusing on possible immunological effects of tungsten exposure.