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The structure of the γ subunit of archaeal translation initiation factor 2 (aIF2) from Sulfolobus solfataricus (SsoIF2γ) was determined in complex with GDPCP (a GTP analog). Crystals were obtained in the absence of magnesium ions in the crystallization solution. They belonged to space group P1, with five molecules in the unit cell. Four of these molecules are related in pairs by a common noncrystallographic twofold symmetry axis, while the fifth has no symmetry equivalent. Analysis of the structure and its comparison with other known aIF2 γ‐subunit structures in the GTP‐bound state show that (i) the magnesium ion is necessary for the formation and the maintenance of the active form of SsoIF2γ and (ii) in addition to the two previously known structural switches 1 and 2, eukaryotic translation initiation factor 2 (eIF2) and aIF2 molecules have another flexible region (switch 3), the function of which may consist of initiation of the hydrolysis of GTP and the removal of e/aIF2 from the ribosome after codon–anticodon recognition. [ABSTRACT FROM AUTHOR]

Regulation of mRNA translation is of vital importance for a cell to adapt to environmental changes. To serve this purpose, intricate mechanisms controlling mRNA translation have evolved, of which the eukaryotic initiation factors eIF2 and eIF4 represent essential regulatory nodes for both stress sensing and signal transduction. Stress sensing by eIF2 α subunit (eIF2α) kinases, translation regulation by eIF2α subunit phosphorylation and subsequent dephosphorylation constitute a core molecular switch for stress adaptation and rapid metabolic regulation. It is not surprising; therefore, that dysfunction of such a pathway is implicated in human disease, especially metabolic syndrome and diabetes. In theory, therapeutic intervention to target the eIF2α phosphorylation pathway provides a promising therapeutic solution to tackle this debilitating syndrome. Careful evaluation of such therapies is crucially needed considering the central role of eIF2α pathway in cellular function for every organ. [ABSTRACT FROM AUTHOR]

Eukaryotic translation initiation factor 2 (eIF2) is a G-protein that functions as a central switch in the initiation of protein synthesis. In its GTP-bound state it delivers the methionyl initiator tRNA (Met-tRNAi) to the small ribosomal subunit and releases it upon GTP hydrolysis following the recognition of the initiation codon. We have developed a complete thermodynamic framework for the assembly of the Saccharomyces cerevisiae eIF2·GTP·Met-tRNAi ternary complex and have determined the effect of the conversion of GTP to GDP on eIF2''s affinity for Met-tRNAi in solution. In its GTP-bound state the factor forms a positive interaction with the methionine moiety on Met-tRNAi that is disrupted when GTP is replaced with GDP, while contacts between the factor and the body of the tRNA remain intact. This positive interaction with the methionine residue on the tRNA may serve to ensure that only charged initiator tRNA enters the initiation pathway. The toggling on and off of the factor''s interaction with the methionine residue is likely to play an important role in the mechanism of initiator tRNA release upon initiation codon recognition. In addition, we show that the conserved base-pair A1:U72, which is known to be a critical identity element distinguishing initiator from elongator methionyl tRNA, is required for recognition of the methionine moiety by eIF2. Our data suggest that a role of this base-pair is to orient the methionine moiety on the initiator tRNA in its recognition pocket on eIF2. [Copyright &y& Elsevier]

The double-stranded (ds) RNA-activated protein kinase (PKR) plays an important role in control of viral infections and cell growth. We have studied the role of PKR in viral infection in mice that are defective in the PKR signaling pathway. Transgenic mice were derived that constitutively express a trans-dominant-negative kinase-defective mutant PKR under control of the β-actin promoter. The trans-dominant-negative PKR mutant expressing transgenic mice do not have a detectable phenotype, similar to observations with PKR knock-out mice. The requirement for PKR in viral pathogenesis was studied by intracerebral infection of mice with a mouse-adapted poliovirus. Histopathological analysis revealed diffuse encephalomyelitis with severe inflammatory lesions throughout the central nervous system (CNS) in infected wild-type mice. In contrast, histopathological evaluation of virus-injected trans-dominant-negative PKR transgenic mice as well as PKR knock-out mice yielded no signs of tissue damage associated with inflammatory host responses. However, the virus did replicate in both models of PKR-deficient mice at a level equal to that observed in wild-type infected mice. Although the results indicate a clear difference in susceptibility to poliovirus-induced encephalitis, this difference manifests clinically as a slight delay in fatal neuropathy in trans-dominant-negative PKR transgenic and PKR knock-out animals. Our observations support the finding that viral-induced PKR activation may play a significant role in pathogenesis by mediating the host response to viral CNS infection. They support PKR to be an effective target to control tissue damage due to deleterious host responses to viral infection. [Copyright &y& Elsevier]

Aim: To study the localization and function of a eukaryotic initiation factor 2 (eIF2α)-associated 67-kDa glycoprotein (p67)., Methods: Immunofluorescence staining, (35)S-Met/Cys metabolic labeling, Western blotting analysis, sucrose gradient centrifugation and high speed centrifugation were used to determine the localization of proteins in transiently transfected COS-1 cells. Transient co-transfection followed by co-immunoprecipitation was used to study the interaction between p67 and double-stranded RNA (dsRNA)-dependent protein kinase (PKR). Wheat germ agglutinin agarose beads were used to absorb glycosylated proteins. In vivo(32)P-labeling followed by immunoprecipitation and Western blotting were used to measure PKR autophosphorylation, eIF2α phosphorylation, and p67 expression in normal and breast cancer cells., Results: The image from immunofluorescence staining showed that p67 was overexpressed in the cytosol but not in the nucleus. In a sucrose gradient, approximately 30% of the overexpressed p67 was bound with ribosomes. p67 interacted with the kinase domain, but not the dsRNA-binding domains of PKR. Only the glycosylated p67 was associated with the ribosome, and p67 did not compete with PKR for ribosome binding. In breast cancer cells, there was increased autophosphorylation of PKR but no phosphorylation of eIF2α, compared with normal breast cells.α The ratio of glycosylated/deglycosylated p67 was altered in breast cancer cells., Conclusion: Glycosylation of p67 is required for its ribosomal association and can potentially inhibit PKR via interaction with the kinase domain of PKR.