New insights into the interactions of the translation initiation factor 2 from archaea with guanine nucleotides and initiator tRNA

Heterotrimeric a/eIF2alphabetagamma (archaeal homologue of the eukaryotic translation initiation factor 2 with alpha, beta and gamma subunits) delivers charged initiator tRNA (tRNAi) to the small ribosomal subunit. In this work, we determined the structures of aIF2gamma from the archaeon Sulfolobus solfataricus in the nucleotide-free and GDP-bound forms. Comparison of the free, GDP and Gpp(NH)p-Mg2+ forms of aIF2gamma revealed a sequence of conformational changes upon GDP and GTP binding. Our results show that the affinity of GDP to the G domain of the gamma subunit is higher than that of Gpp(NH)p. In analyzing a pyrophosphate molecule binding to domain II of the gamma subunit, we found a cleft that is very suitable for the acceptor stem of tRNA accommodation. It allows the suggestion of an alternative position for Met-tRNA i Met on the alphagamma intersubunit dimer, at variance with a recently published one. In the model reported here, the acceptor stem of the tRNAi is approximately perpendicular to that of tRNA in the ternary complex elongation factor Tu-Gpp(NH)p-tRNA. According to our analysis, the elbow and T stem of Met-tRNA i Met in this position should make extensive contact with the alpha subunit of aIF2. Thus, this model is in good agreement with experimental data showing that the alpha subunit of aIF2 is necessary for the stable interaction of aIF2gamma with Met-tRNA i Met.     

Thermodynamic analysis reveals that GTP binding affects the interaction between the alpha- and gamma-subunits of translation initiation factor 2

Eukaryotic and archaeal translation initiation factors 2, heterotrimers that consist of α-, β-, and γ-subunits, deliver methionylated initiator tRNA to a small ribosomal subunit in a manner that depends on GTP. To evaluate correlation of the function and association of the subunits, we used isothermal titration calorimetry to analyze the thermodynamics of the interactions between the α- and γ-subunits in the presence or absence of a nonhydrolyzable GTP analog or GDP. The α-subunits bound to the γ-subunit with large heat capacity change (ΔC_p) values. The ΔH and ΔC_p values for the interaction between the α- and γ-subunits varied in the presence of the GTP analog but not in the presence of GDP. These results suggest that the binding of both the α-subunit and GTP changes the conformation of the switch region of the γ-subunit and increases the affinity of the γ-subunit for tRNA.     

Structure of the archaeal translation initiation factor aIF2 beta from Methanobacterium thermoautotrophicum: implications for translation initiation

aIF2 beta is the archaeal homolog of eIF2 beta, a member of the eIF2 heterotrimeric complex, implicated in the delivery of Met-tRNA(i)(Met) to the 40S ribosomal subunit. We have determined the solution structure of the intact beta-subunit of aIF2 from Methanobacterium thermoautotrophicum. aIF2 beta is composed of an unfolded N terminus, a mixed alpha/beta core domain and a C-terminal zinc finger. NMR data shows the two folded domains display restricted mobility with respect to each other. Analysis of the aIF2 gamma structure docked to tRNA allowed the identification of a putative binding site for the beta-subunit in the ternary translation complex. Based on structural similarity and biochemical data, a role for the different secondary structure elements is suggested.     

Translation initiation complex formation in the crenarchaeon Sulfolobus solfataricus

The function of initiation factors in and the sequence of events during translation initiation have been intensively studied in Bacteria and Eukaryotes, whereas in Archaea knowledge on these functions/processes is limited. By employing chemical probing, we show that translation initiation factor aIF1 of the model crenarchaeon Sulfolobus solfataricus binds to the same area on the ribosome as the bacterial and eukaryal orthologs. Fluorescence energy transfer assays (FRET) showed that aIF1, like its eukaryotic and bacterial orthologs, has a fidelity function in translation initiation complex formation, and that both aIF1 and aIF1A exert a synergistic effect in stimulating ribosomal association of the Met-tRNAi^Met binding factor a/eIF2. However, as in Eukaryotes their effect on a/eIF2 binding appears to be indirect. Moreover, FRET was used to analyze for the first time the sequence of events toward translation initiation complex formation in an archaeal model system. These studies suggested that a/eIF2-GTP binds first to the ribosome and then recruits Met-tRNAi^Met, which appears to comply with the operational mode of bacterial IF2, and deviates from the shuttle function of the eukaryotic counterpart eIF2. Thus, despite the resemblance of eIF2 and a/eIF2, recruitment of initiator tRNA to the ribosome is mechanistically different in Pro- and Eukaryotes.     

Crystal structure of the C-terminal domain of the ϵ subunit of human translation initiation factor eIF2B

Eukaryotic translation initiation factor eIF2B, the guanine nucleotide exchange factor (GEF) for eIF2, catalyzes conversion of eIF2·GDP to eIF2·GTP. The eIF2B is composed of five subunits, α, β, γ, δ and ε, within which the ε subunit is responsible for catalyzing the guanine exchange reaction. Here we present the crystal structure of the C-terminal domain of human eIF2Bε (eIF2Bε-CTD) at 2.0-Å resolution. The structure resembles a HEAT motif and three charge-rich areas on its surface can be identified. When compared to yeast eIF2Bε-CTD, one area involves highly conserved AA boxes while the other two are only partially conserved. In addition, the previously reported mutations in human eIF2Bε-CTD, which are related to the loss of the GEF activity and human VWM disease, have been discussed. Based on the structure, most of such mutations tend to destabilize the HEAT motif.     

Isolation and crystallization of heterotrimeric translation initiation factor 2 from <Emphasis Type="Italic">Sulfolobus solfataricus</Emphasis>

The structure of the intact heterotrimeric translation initiation factor 2 (e/aIF2) is of great interest due to its key role in the initiator tRNA delivery to the ribosome and in translation initiation regulation in eukaryotes and archaea. We have chosen aIF2 from the hyperthermophilic archaeobacterium Sulfolobus solfataricus (SsoIF2) as an object for crystallization and structural investigations. Genes of the SsoIF2 subunits α, β, and γ were cloned and superexpressed. A method for heterotrimer SsoIF2αβγ purification was elaborated with at least 95% purity. Highly ordered crystals of the full-sized SsoIF2, reflecting X-rays at the resolution up to 2.8 Å, were obtained for the first time.     

Stepwise assembly of the eukaryotic translation initiation factor 2 complex

The eukaryotic translation initiation factor 2 (eIF2) has key functions in the initiation step of protein synthesis. eIF2 guides the initiator tRNA to the ribosome, participates in scanning of the mRNA molecule, supports selection of the start codon, and modulates the translation of mRNAs in response to stress. eIF2 comprises a heterotrimeric complex whose assembly depends on the ATP-grasp protein Cdc123. Mutations of the eIF2γ subunit that compromise eIF2 complex formation cause severe neurological disease in humans. To this date, however, details about the assembly mechanism, step order, and the individual functions of eIF2 subunits remain unclear. Here, we quantified assembly intermediates and studied the behavior of various binding site mutants in budding yeast. Based on these data, we present a model in which a Cdc123-mediated conformational change in eIF2γ exposes binding sites for eIF2α and eIF2β subunits. Contrary to an earlier hypothesis, we found that the associations of eIF2α and eIF2β with the γ-subunit are independent of each other, but the resulting heterodimers are nonfunctional and fail to bind the guanosine exchange factor eIF2B. In addition, levels of eIF2α influence the rate of eIF2 assembly. By binding to eIF2γ, eIF2α displaces Cdc123 and thereby completes the assembly process. Experiments in human cell culture indicate that the mechanism of eIF2 assembly is conserved between yeast and humans. This study sheds light on an essential step in eukaryotic translation initiation, the dysfunction of which is linked to human disease.     

Characterization of plant eukaryotic translation initiation factor 6 (eIF6) genes: The essential role in embryogenesis and their differential expression in Arabidopsis and rice

Eukaryotic translation initiation factor 6 (eIF6) is an essential component of ribosome biogenesis. In our present study, we characterize plant eIF6 genes for the first time. Although a single gene encodes eIF6 in yeast and animals, two genes were found to encode proteins homologous to animal and yeast eIF6 in Arabidopsis and rice, denoted At-eIF6;1 and At-eIF6;2, and Os-eIF6;1 and Os-eIF6;2, respectively. Analysis of the yeast eif6 (tif6) mutant suggested that plant eIF6, at least in the case of At-eIF6;1, can complement the essential function of eIF6 in yeast. Evidence for the essential role of eIF6 in plants was also provided by the embryonic-lethal phenotype of the at-eif6;1 mutant. In contrast, At-eIF6;2 appears not to be essential due to its very low expression level and the normal growth phenotype of the eif6;2 mutants. Consistent with the putative role of plant eIF6 in ribosome biogenesis, At-eIF6;1 is predominately expressed in tissues where cell division actively proceeds under the control of intronic cis-regulatory elements. On the other hand, both Os-eIF6;1 and Os-eIF6;2 are probably active genes because they are expressed at significant expression levels. Interestingly, the supply of ammonium nitrate as a plant nutrient was found to induce specifically the expression of Os-eIF6;2. Our present findings indicate that the eIF6 genes have differently evolved in plant and animal kingdoms and also in distinct plant species.     

Structure of translation initiation factor 1 from Mycobacterium tuberculosis and inferred binding to the 30S ribosomal subunit

The crystal structure of the free form of IF1 from Mycobacterium tuberculosis has been determined at 1.47 Å resolution. The structure adopts the expected OB fold and matches the high structural conservation among IF1 orthologues. In order to further explore the function of Mtb-IF1, we built a model of its interaction with the 30S ribosomal subunit based on the crystal structure of the complex from Thermus thermophilus. The model suggests that several functionally important side chain residues undergo large movements while the rest of the protein in complex shows only very limited conformational change as compared to its form in solution.     

The crystal structure of the N-terminal region of the alpha subunit of translation initiation factor 2 (eIF2alpha) from Saccharomyces cerevisiae provides a view of the loop containing serine 51, the target of the eIF2alpha-specific kinases

The alpha subunit of translation initiation factor 2 (eIF2alpha) is the target of specific kinases that can phosphorylate a conserved serine residue as part of a mechanism for regulating protein expression at the translational level in eukaryotes. The structure of the 20 kDa N-terminal region of eIF2alpha from Saccharomyces cerevisiae was determined by X-ray crystallography at 2.5A resolution. In most respects, the structure is similar to that of the recently solved human eIF2alpha; the rather elongated protein contains a five-stranded antiparallel beta-barrel in its N-terminal region, followed by an almost entirely helical domain. The S.cerevisiae eIF2alpha lacks a disulfide bridge that is present in the homologous protein in humans and some of the other higher eukaryotes. Interestingly, a conserved loop consisting of residues 51-65 and containing serine 51, the putative phosphorylation site, is visible in the electron density maps of the S.cerevisiae eIF2alpha; most of this functionally important loop was not observed in the crystal structure of the human protein. This loop is relatively exposed to solvent, and contains two short 3(10) helices in addition to some extended structure. Serine 51 is located at the C-terminal end of one of the 3(10) helices and near several conserved positively charged residues. The side-chain of serine 51 is sufficiently exposed so that its phosphorylation would not necessitate a substantial change in the protein structure. The structures and relative positions of residues that have been implicated in kinase binding and in the interaction with guanine nucleotide exchange factor (eIF2B) are described.     

Identification of MRI1, encoding translation initiation factor eIF-2B subunit alpha/beta/delta-like protein,as a candidate locus for infantile epilepsy with severe cystic degeneration of the brain

Several neurodegenerative disorders are known to predominantly affect the white matter of the brain including vanishing white matter disease (VWMD), an autosomal recessive disorder characterized by leukodystrophy of varying severity in addition to variable systemic involvement. We report a consanguineous Arab family with three affected children, all of whom presented with severe neonatal epilepsy and profound neurodegenerative disease characterized by marked leukodystrophy with white matter cavitation mimicking VWMD. We combined autozygome and exome analysis to identify a novel variant in the gene encoding a member of the eIF2B-related family of proteins (MRI1). This is a poorly understood family of proteins of unclear function. Our results represent the first link between a variant in a member of this family and a human disease, and suggest that it converges with the highly homologous eIF2B, known to be mutated in VWMD, on the molecular pathogenesis of neurodegeneration.     

Crystal structure of the regulatory subunit of archaeal initiation factor 2B (aIF2B) from hyperthermophilic archaeon Pyrococcus horikoshii OT3: a proposed structure of the regulatory subcomplex of eukaryotic IF2B

Eukaryotic translation initiation factor 2B (eIF2B) is the guanine-nucleotide exchange factor for eukaryotic initiation factor 2 (eIF2). eIF2B is a heteropentameric protein composed of alpha- subunits. The alpha, beta, and delta subunits form a regulatory subcomplex, while the gamma and form a catalytic subcomplex. Archaea possess homologues of alpha, beta, and delta subunits of eIF2B. Here, we report the three-dimensional structure of an archaeal regulatory subunit (aIF2Balpha) from the hyperthermophilic archaeon Pyrococcus horikoshii OT3 determined by X-ray crystallography at 2.2A resolution. aIF2Balpha consists of two subdomains, an N-domain (residues 1-95) and a C-domain (residues 96-276), connected by a long alpha-helix (alpha5: 78-106). The N-domain contains a five helix bundle structure, while the C-domain folds into the alpha/beta structure, thus showing similarity to D-ribose-5-phosphate isomerase structure. The presence of two molecules in the crystallographic asymmetric unit and the gel filtration analysis suggest a dimeric structure of aIF2Balpha in solution, interacting with each other by C-domains. Furthermore, the crystallographic 3-fold symmetry generates a homohexameric structure of aIF2Balpha; the interaction is primarily mediated by the long alpha-helix at the N-domains. This structure suggests an architecture of the three subunits, alpha, beta, and delta, in the regulatory subcomplex within eIF2B.     

The stability of eukaryotic initiation factor 2-associated glycoprotein, p67, increases during skeletal muscle differentiation and that inhibits the phosphorylation of extracellular signal-regulated kinases 1 and 2

Eukaryotic initiation factor 2-associated glycoprotein, p67, protects eIF2 from phosphorylation by its kinases. To understand the roles of p67 during skeletal muscle differentiation of mouse C2C12 myoblasts, we measured the level of p67 during myotube formation. We noticed that the level of p67 increases during myoblast differentiation and this increased level is controlled at the translational stage. The stability of p67 in the myotubes is due to its low turnover rate. The phosphorylation of the extracellular signal-regulated kinases (ERKs 1 and 2) is high in growth-factor-mediated cycling of C2C12 myoblasts and this phosphorylation decreases at 96 h when these myoblasts are grown in differentiation medium. At this time of differentiation, the level of p67 is higher compared to 0 h of differentiation. p67 binds to ERK2 and inhibits its activity in vitro. Taken together, these results suggest that the stability of p67 increases during myotube formation while inhibiting the phosphorylation of ERKs 1 and 2.     

Eukaryotic translation initiation factor 5A induces apoptosis in colon cancer cells and associates with the nucleus in response to tumour necrosis factor alpha signalling

Eukaryotic translation initiation factor 5A (eIF5A) is thought to function as a nucleocytoplasmic shuttle protein. There are reports of its involvement in cell proliferation, and more recently it has also been implicated in the regulation of apoptosis. In the present study, we examined the effects of eIF5A over-expression on apoptosis and of siRNA-mediated suppression of eIF5A on expression of the tumour suppressor protein, p53. Over-expression of either eIF5A or a mutant of eIF5A incapable of being hypusinated was found to induce apoptosis in colon carcinoma cells. Our results also indicate that eIF5A is required for expression of p53 following the induction of apoptosis by treatment with Actinomycin D. Depiction of eIF5A localization by indirect immunofluorescence has indicated, for the first time, that the protein is rapidly translocated from the cytoplasm to the nucleus by death receptor activation or following treatment with Actinomycin D. These findings collectively indicate that unhypusinated eIF5A may have pro-apoptotic functions and that eIF5A is rapidly translocated to the nucleus following the induction of apoptotic cell death.     

Selective involvement of members of the eukaryotic initiation factor 4E family in the infection of Arabidopsis thaliana by potyviruses

Arabidopsis thaliana plants with mutations in the genes encoding eukaryotic initiation factor (eIF4E) or isoform of eIF4E (eIF(iso)4E) were tested for susceptibility to Clover yellow vein virus (ClYVV), a member of the genus Potyvirus. ClYVV accumulated in both inoculated and upper uninoculated leaves of mutant plants lacking eIF(iso)4E, but not in mutant plants lacking eIF4E. In contrast, Turnip mosaic virus (TuMV), another member of the genus Potyvirus, multiplied in mutant plants lacking eIF4E but not in mutant plants lacking eIF(iso)4E. These results suggest the selective involvement of members of the eIF4E family in infection by potyviruses.     

Mitochondrial Sulfide Quinone Oxidoreductase Prevents Activation of the Unfolded Protein Response in Hydrogen Sulfide

Hydrogen sulfide (H₂S) is an endogenously produced gaseous molecule with important roles in cellular signaling. In mammals, exogenous H₂S improves survival of ischemia/reperfusion. We have previously shown that exposure to H₂S increases the lifespan and thermotolerance in Caenorhabditis elegans, and improves protein homeostasis in low oxygen. The mitochondrial SQRD-1 (sulfide quinone oxidoreductase) protein is a highly conserved enzyme involved in H₂S metabolism. SQRD-1 is generally considered important to detoxify H₂S. Here, we show that SQRD-1 is also required to maintain protein translation in H₂S. In sqrd-1 mutant animals, exposure to H₂S leads to phosphorylation of eIF2α and inhibition of protein synthesis. In contrast, global protein translation is not altered in wild-type animals exposed to lethally high H₂S or in hif-1(ia04) mutants that die when exposed to low H₂S. We demonstrate that both gcn-2 and pek-1 kinases are involved in the H₂S-induced phosphorylation of eIF2α. Both ER and mitochondrial stress responses are activated in sqrd-1 mutant animals exposed to H₂S, but not in wild-type animals. We speculate that SQRD-1 activity in H₂S may coordinate proteostasis responses in multiple cellular compartments.     

Change in nutritional status modulates the abundance of critical pre-initiation intermediate complexes during translation initiation in vivo

In eukaryotic translation initiation, eIF2GTP-Met-tRNA(i)(Met) ternary complex (TC) interacts with eIF3-eIF1-eIF5 complex to form the multifactor complex (MFC), while eIF2GDP associates with eIF2B for guanine nucleotide exchange. Gcn2p phosphorylates eIF2 to inhibit eIF2B. Here we evaluate the abundance of eIFs and their pre-initiation intermediate complexes in gcn2 deletion mutant grown under different conditions. We show that ribosomes are three times as abundant as eIF1, eIF2 and eIF5, while eIF3 is half as abundant as the latter three and hence, the limiting component in MFC formation. By quantitative immunoprecipitation, we estimate that approximately 15% of the cellular eIF2 is found in TC during rapid growth in a complex rich medium. Most of the TC is found in MFC, and important, approximately 40% of the total eIF2 is associated with eIF5 but lacks tRNA(i)(Met). When the gcn2Delta mutant grows less rapidly in a defined complete medium, TC abundance increases threefold without altering the abundance of each individual factor. Interestingly, the TC increase is suppressed by eIF5 overexpression and Gcn2p expression. Thus, eIF2B-catalyzed TC formation appears to be fine-tuned by eIF2 phosphorylation and the novel eIF2/eIF5 complex lacking tRNA(i)(Met).     

Eukaryotic initiation factor 2alpha kinase is a nitric oxide-responsive mercury sensor enzyme: potent inhibition of catalysis by the mercury cation and reversal by nitric oxide

The activity of one of the eukaryotic initiation factor 2alpha kinases, heme-regulated inhibitor (HRI), is modulated by heme binding. Here, we demonstrate for the first time that Hg2+ strongly inhibits the function of HRI (IC50=0.6 microM), and nitric oxide fully reverses this inhibition. Other divalent metal cations, such as Fe2+, Cu2+, Cd2+, Zn2+ and Pb2+, also significantly inhibit kinase activity with IC50 values of 1.9-8.5 microM. Notably, inhibition by cations other than Hg2+ is not reversed by nitric oxide. Our present data support dual roles of Hg2+ and nitric oxide in the regulation of protein synthesis during cell emergency states.