Universally conserved translation initiation factors

The process by which translation is initiated has long been considered similar in Bacteria and Eukarya but accomplished by a different unrelated set of factors in the two cases. This not only implies separate evolutionary histories for the two but also implies that at the universal ancestor stage, a translation initiation mechanism either did not exist or was of a different nature than the extant processes. We demonstrate herein that (i) the "analogous" translation initiation factors IF-1 and eIF-1A are actually related in sequence, (ii) the "eukaryotic" translation factor SUI1 is universal in distribution, and (iii) the eukaryotic/archaeal translation factor eIF-5A is homologous to the bacterial translation factor EF-P. Thus, the rudiments of translation initiation would seem to have been present in the universal ancestor stage. However, significant development and refinement subsequently occurred independently on both the bacterial lineage and on the archaeal/eukaryotic line.     

Mutations in the alpha subunit of eukaryotic translation initiation factor 2 (eIF-2 alpha) that overcome the inhibitory effect of eIF-2 alpha phosphorylation on translation initiation

Phosphorylation of eIF-2 alpha in Saccharomyces cerevisiae by the protein kinase GCN2 leads to inhibition of general translation initiation and a specific increase in translation of GCN4 mRNA. We isolated mutations in the eIF-2 alpha structural gene that do not affect the growth rate of wild-type yeast but which suppress the toxic effects of eIF-2 alpha hyperphosphorylation catalyzed by mutationally activated forms of GCN2. These eIF-2 alpha mutations also impair translational derepression of GCN4 in strains expressing wild-type GCN2 protein. All four mutations alter single amino acids within 40 residues of the phosphorylation site in eIF-2 alpha; however, three alleles do not decrease the level of eIF-2 alpha phosphorylation. We propose that these mutations alter the interaction between eIF-2 and its recycling factor eukaryotic translation initiation factor 2B (eIF-2B) in a way that diminishes the inhibitory effect of phosphorylated eIF-2 on the essential function of eIF-2B in translation initiation. These mutations may identify a region in eIF-2 alpha that participates directly in a physical interaction with the GCN3 subunit of eIF-2B.     

Removal of beta subunit of the eukaryotic polypeptide chain initiation factor 2 by limited proteolysis

It is generally considered that the eukaryotic polypeptide chain initiation factor 2 (eIF-2) from rabbit reticulocytes consists of three nonidentical subunits termed alpha, beta, and gamma, in order of increasing molecular weight. However, a recent report [Stringer, E. A., Chaudhuri, A., Valenzuela, D. & Maitra, U. (1980) Proc. Natl. Acad. Sci. USA 77, 3356-3359] suggested that this factor is made up of only two subunits. In this paper we show that limited proteolysis of rabbit reticulocyte eIF-2 leads to loss of the beta subunit. This modified eIF-2 has the same activity as the native factor in promoting ternary (eIF-2.GTP.Met-tRNAi) and 40S (eIF-2.GTP.Met-tRNAi.40S ribosome) initiation complex formation. Like native eIF-2, the protease-treated factor can restore translation in heme-deficient lysates. On the other hand, the treated factor is less stable than the native protein.     

Use of monoclonal antibodies to study the structure and function of eukaryotic protein synthesis initiation factor eIF-2B

The eukaryotic protein synthesis initiation factor, eIF-2B, is a multimeric protein of five different subunits termed alpha, beta, gamma, delta and epsilon, which facilitates recycling of a further factor, eIF-2, and is an important control point in the initiation process. In order to investigate the structure and function of eIF-2B, monoclonal antibodies have been prepared to the beta, delta and epsilon subunits of the factor from rabbit reticulocytes. All three antibodies are active in Western blotting, ELISA and immunoprecipitation. The anti-epsilon antibody inhibits both the guanine nucleotide exchange activity of eIF-2B and protein synthesis in the rabbit reticulocyte lysate at the level of initiation. The other two antibodies do not inhibit either guanine nucleotide exchange or protein synthesis. The monoclonal antibodies and a polyclonal anti-(rabbit reticulocyte eIF-2B) serum were used to investigate the subunit size and the antigenic structure of eIF-2B from a variety of rabbit tissues and from a variety of mammalian species. eIF-2B from all rabbit tissues tested was indistinguishable from that prepared from rabbit reticulocytes. Quantitative studies showed substantial variation in the relative concentrations of eIF-2 and eIF-2B between different rabbit tissues. Marked variation in both the sizes of the subunits and their reaction with the antibodies was observed between eIF-2B from rabbit, rat, guinea pig and man.     

Ligand interactions with eukaryotic translation initiation factor 2: role of the gamma-subunit

Eukaryotic translation initiation factor 2 (eIF-2) comprises three non-identical subunits alpha, beta and gamma. In vitro, eIF-2 binds the initiator methionyl-tRNA in a GTP-dependent fashion. Based on similarities between eukaryotic eIF-2gamma proteins and eubacterial EF-Tu proteins, we previously proposed a major role for the gamma-subunit in binding guanine nucleotide and tRNA. We have tested this hypothesis by examining the biochemical activities of yeast eIF-2 purified from wild-type strains and strains harboring mutations in the eIF-2gamma structural gene (GCD11) predicted to alter ligand binding by eIF-2. The alteration of tyrosine 142 in yeast eIF-2gamma, corresponding to histidine 66 in Escherichia coli EF-Tu, dramatically reduced the affinity of eIF-2 for Met-tRNAi(Met) without affecting the k(off) value for guanine nucleotides. In contrast, non-lethal substitutions at a conserved lysine residue (K250) in the putative guanine ring-binding loop increased the off-rate for GDP, thereby mimicking the function of the guanine nucleotide exchange factor eIF-2B, without altering the apparent dissociation constant for Met-tRNAi(Met). For eIF-2[gamma-K250R], the increased off-rate also seen for GTP was masked by the presence of Met-tRNAi(Met) in vitro. In vivo, increasing the dose of the yeast initiator tRNA gene suppressed the slow-growth phenotype and reduced GCN4 expression in gcd11-K250R and gcd11-Y142H strains. These studies indicate that the gamma-subunit of eIF-2 does indeed provide EF-Tu-like function to the eIF-2 complex, and further suggest that the level of Met-tRNAi(Met) is critical for maintaining wild-type rates of initiation in vivo.     

Identification of cDNA clones for the large subunit of eukaryotic translation initiation factor 3. Comparison of homologues from human, Nicotiana tabacum, Caenorhabditis elegans, and Saccharomyces cerevisiae

Initiation of translation in eukaryotes is mediated by a set of initiation factors. Mammalian initiation factor 3 is composed of at least 8 subunits, with the largest being about 180 kDa in size. Here we report the cloning of the p180 subunit of human eukaryotic translation initiation factor (eIF) 3. The amino acid sequence deduced from the cDNA agrees with the sequences of CNBr fragments of eIF-3, confirming the identity of the clone. The 1382 amino acid open reading frame contains a high percentage of charged residues (48%) and an unusual repetitive domain near the carboxyl terminus composed of 25 repeats of 10 amino acids each. Data base searches identified related sequences found in members of the plant and fungal kingdoms as well as in other mammals and the nematode Caenorhabditis elegans. These sequences share significant identity with the human clone and probably represent the homologues of the p180 subunit in these organisms. This is the first report identifying the sequence of the large subunit of eIF-3.     

The alpha-subunit of the mammalian guanine nucleotide-exchange factor eIF-2B is essential for catalytic activity in vitro

Eukaryotic initiation factor (eIF)-2B, the guanine nucleotide exchange factor for eIF-2, consists of five distinct subunits in both mammals and the yeast Saccharomyces cerevisiae. The exchange reaction mediated by eIF-2B can be regulated by phosphorylation of eIF-2 on its alpha-subunit. This represents a key control point in the initiation of translation. The functions of the individual subunits of the eIF-2B complex remain unclear. Mutational analysis in Saccharomyces cerevisiae suggested that the smallest subunit (the alpha) is dispensable for exchange, but required for the inhibition of eIF-2B by eIF-2(alphaP). Here we present evidence that, in mammalian cells, eIF-2Balpha is essential for the activity of the complex, since preparations of eIF-2B lacking this subunit are not active in nucleotide exchange in vitro, although the complex still contains the beta, gamma, delta and epsilon subunits.     

A difference in the rate of ribosomal elongation balances the synthesis of eukaryotic translation initiation factor (eIF)-2 alpha and eIF-2 beta

Eukaryotic translation initiation factor 2 (eIF-2) is a heterotrimer composed of three subunits designated alpha, beta, and gamma. These proteins exist in equimolar amounts in the cell and have not been detected as isolated subunits. Our research examines the basis of their balanced synthesis. Northern analysis of K562 cell mRNA revealed that eIF-2 beta was five times more abundant than eIF-2 alpha. However, immunoprecipitation of pulse-labeled K562 cells showed an equimolar rate of synthesis of eIF-2 alpha and -beta despite the 5-fold difference in the size of their mRNA pools. Addition of equal amounts of synthetic capped mRNA for eIF-2 alpha and eIF-2 beta to an in vitro translation reaction produced five times more eIF-2 alpha protein than eIF-2 beta. Determination of the polysome profile for alpha and beta mRNA in K562 cells indicated eIF-2 alpha was translated more efficiently than eIF-2 beta. Substitution of either the initiation codon context or the leader of the beta mRNA for that of alpha had only a minor effect on the translational efficiency of beta. Comparison of the rate of ribosomal elongation for the two mRNAs indicated that ribosomes associated with the beta mRNA elongate at a rate 4-fold less than that of eIF-2 alpha. Thus, the balanced translation of alpha and beta mRNA is primarily the result of a 4-fold difference in the rate of ribosomal elongation.     

Localization of eukaryotic initiation factor 2 in neuron primary cultures and established cell lines

Eukaryotic initiation factor 2 (eIF-2) is a heterotrimeric protein with subunits alpha, beta and gamma that forms a ternary complex with Met-tRNA and GTP. It promotes the binding of Met-tRNA to ribosomes and controls translational rates via phosphorylation/dephosphorylation mechanisms. By means of immunofluorescence and post-embedding immunocytochemistry of intact cells and quantitative immunoblotting of cell extracts, the cellular distribution of the initiation factor has been examined in primary neuronal cultures as well as in two established cell lines: PC12 phaeochromocytoma cells and rat pituitary GH4C1 cells. Our results indicated that the initiation factor is located not only in the cytoplasm but also in the nuclei of the cultured neurons and cell lines. In the cytoplasm, immunocytochemical studies reveal that the factor is present mainly in those areas that are rich in ribosomes. In the nucleus, the immunolabelling of eukaryotic initiation factor 2 verified the presence of gold particles in both nucleolar and extranucleolar areas. The specific distribution of this factor on both sides of the nuclear envelope suggests that it might have some nuclear-related function(s) besides its already known role in the control of translation.     

Phosphorylation of plant eukaryotic initiation factor-2 by the plant-encoded double-stranded RNA-dependent protein kinase, pPKR, and inhibition of protein synthesis in vitro

Regulation of protein synthesis by eukaryotic initiation factor-2alpha (eIF-2alpha) phosphorylation is a highly conserved phenomenon in eukaryotes that occurs in response to various stress conditions. Protein kinases capable of phosphorylating eIF-2alpha have been characterized from mammals and yeast. However, the phenomenon of eIF2-alpha-mediated regulation of protein synthesis and the presence of an eIF-2alpha kinase has not been demonstrated in higher plants. We show that plant eIF-2alpha (peIF-2alpha) and mammalian eIF-2alpha (meIF-2alpha) are phosphorylated similarly by both the double-stranded RNA-binding kinase, pPKR, present in plant ribosome salt wash fractions and the meIF-2alpha kinase, PKR. By several criteria, phosphorylation of peIF-2alpha is directly correlated with pPKR protein and autophosphorylation levels. Significantly, pPKR is capable of specifically phosphorylating Ser51 in a synthetic eIF-2alpha peptide, a key characteristic of the eIF-2alpha kinase family. Taken together, these data support the concept that pPKR is a member of the eIF-2alpha kinase family. In addition, the inhibition of brome mosaic virus RNA in vitro translation in wheat germ lysates by the addition of double-stranded RNA, phosphorylated peIF-2alpha, meIF-2alpha, or activated human PKR suggests that plant protein synthesis may be regulated via phosphorylation of eIF-2alpha.     

Expression of a phosphorylation-resistant eukaryotic initiation factor 2 alpha-subunit mitigates heat shock inhibition of protein synthesis

Protein synthesis is dramatically reduced upon exposure of cells to elevated temperature. Concordant with this inhibition, multiple phosphorylation and dephosphorylation reactions occur on specific eukaryotic initiation factors that are required for protein synthesis. Most notably, phosphorylation of the alpha-subunit of eukaryotic initiation factor-2 (eIF-2 alpha) on serine residue 51 occurs. To identify the importance of phosphorylation in control of protein synthesis, we have evaluated the effects of expression of a mutant eIF-2 alpha which is resistant to phosphorylation. Expression of a serine to alanine mutant at residue 51 of eIF-2 alpha partially protected cells from the inhibition of protein synthesis in response to heat treatment. The overexpressed serine to alanine 51 mutant subunit was incorporated into the eIF-2 heterotrimer and was resistant to phosphorylation. These results are consistent with the hypothesis that heat shock inhibition of translation is mediated in part through phosphorylation of eIF-2 alpha. Expression of the wild type or mutant eIF-2 alpha did not affect cell survival or induction of hsp70 mRNA upon heat shock, indicating that although eIF-2 alpha is a heat shock-induced protein, its increased synthesis during heat shock does not alter the heat-shock response.     

Identification and characterization of pancreatic eukaryotic initiation factor 2 alpha-subunit kinase, PEK, involved in translational control

In response to various environmental stresses, eukaryotic cells down-regulate protein synthesis by phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (eIF-2alpha). In mammals, the phosphorylation was shown to be carried out by eIF-2alpha kinases PKR and HRI. We report the identification and characterization of a cDNA from rat pancreatic islet cells that encodes a new related kinase, which we term pancreatic eIF-2alpha kinase, or PEK. In addition to a catalytic domain with sequence and structural features conserved among eIF-2alpha kinases, PEK contains a distinctive amino-terminal region 550 residues in length. Using recombinant PEK produced in Escherichia coli or Sf-9 insect cells, we demonstrate that PEK is autophosphorylated on both serine and threonine residues and that the recombinant enzyme can specifically phosphorylate eIF-2alpha on serine-51. Northern blot analyses indicate that PEK mRNA is expressed in all tissues examined, with highest levels in pancreas cells. Consistent with our mRNA assays, PEK activity was predominantly detected in pancreas and pancreatic islet cells. The regulatory role of PEK in protein synthesis was demonstrated both in vitro and in vivo. The addition of recombinant PEK to reticulocyte lysates caused a dose-dependent inhibition of translation. In the Saccharomyces model system, PEK functionally substituted for the endogenous yeast eIF-2alpha kinase, GCN2, by a process requiring the serine-51 phosphorylation site in eIF-2alpha. We also identified PEK homologs from both Caenorhabditis elegans and the puffer fish Fugu rubripes, suggesting that this eIF-2alpha kinase plays an important role in translational control from nematodes to mammals.     

A eukaryotic translation initiation factor 2-associated 67 kDa glycoprotein partially reverses protein synthesis inhibition by activated double-stranded RNA-dependent protein kinase in intact cells

A eukaryotic translation initiation factor 2 (eIF-2)-associated 67 kDa glycoprotein (p67) protects the eIF-2 alpha-subunit from inhibitory phosphorylation by eIF-2 kinases, and this promotes protein synthesis in the presence of active eIF-2 alpha kinases in vitro [Ray, M. K., et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 539-543]. We have now examined the effect of overexpression of this cellular eIF-2 kinase inhibitor in an in vivo system using transiently transfected COS-l cells. In this system, coexpression of genes that inhibit PKR activity restores translation of plasmid-derived mRNA. We now report the following. (1) Transient transfection of COS-1 cells with a p67 expression vector increased p67 synthesis by 20-fold over endogenous levels in the isolated subpopulation of transfected cells. (2) Cotransfection of p67 cDNA increased translation of plasmid-derived mRNAs. (3) Overexpression of p67 reduced phosphorylation of coexpressed eIF-2 alpha. (4) p67 synthesis was inhibited by cotransfection with an eIF-2 alpha mutant S51D, a mutant that mimics phosphorylated eIF-2 alpha, indicating that p67 cannot bypass translational inhibition mediated by phosphorylation of the eIF-2 alpha-subunit. These results show that the cellular protein p67 can reverse PKR-mediated translational inhibition in intact cells.     

Subunit assembly and guanine nucleotide exchange activity of eukaryotic initiation factor-2B expressed in Sf9 cells

Eukaryotic initiation factor-2B (eIF-2B) is a guanine nucleotide exchange factor (GEF) that plays a key role in the regulation of protein synthesis. In this study, we have used the baculovirus-infected Sf9 insect cell system to express and characterize the five dissimilar subunits of rat eIF-2B. GEF activity was detected in extracts of Sf9 cells expressing the epsilon-subunit alone and was greatly increased when all five subunits were coexpressed. In addition, high GEF activity was observed in extracts containing a four-subunit complex lacking the alpha-subunit. Assembly of an eIF-2B holoprotein was confirmed by coimmunoprecipitation of all five subunits. Gel filtration chromatography revealed that recombinant eIF-2B had the same molecular mass as eIF-2B purified from rat liver and that it did indeed possess GEF activity. Phosphorylation of the substrate eIF-2 inhibited the GEF activity of the five-subunit eIF-2B; this inhibition required the eIF-2B alpha-subunit. The results demonstrate that eIF-2Balpha functions as a regulatory subunit that is not required for GEF activity, but instead mediates the regulation of eIF-2B by substrate phosphorylation. Furthermore, eIF-2Bepsilon is necessary and is perhaps sufficient for GEF activity in vitro.     

Ribosome-binding domain of eukaryotic initiation factor-2 kinase GCN2 facilitates translation control

A family of protein kinases regulate translation initiation in response to cellular stresses by phosphorylation of eukaryotic initiation factor-2 (eIF-2). One family member from yeast, GCN2, contains a region homologous to histidyl-tRNA synthetases juxtaposed to the kinase catalytic domain. It is thought that uncharged tRNA accumulating during amino acid starvation binds to the synthetase-related sequences and stimulates phosphorylation of the alpha subunit of eIF-2. In this report, we define another domain in GCN2 that functions to target the kinase to ribosomes. A truncated version of GCN2 containing only amino acid residues 1467 to 1590 can independently associate with the translational machinery. Interestingly, this region of GCN2 shares sequence similarities with the core of the double-stranded RNA-binding domain (DRBD). Substitutions of the lysine residues conserved among DRBD sequences block association of GCN2 with ribosomes and impaired the ability of the kinase to stimulate translational control in response to amino acid limitation. Additionally, as found for other DRBD sequences, recombinant protein containing GCN2 residues 1467-1590 can bind double-stranded RNA in vitro, suggesting that interaction with rRNA mediates ribosome targeting. These results indicate that appropriate ribosome localization of the kinase is an obligate step in the mechanism leading to translational control by GCN2.     

Initiation factor eIF-4E of Saccharomyces cerevisiae. Distribution within the cell, binding to mRNA, and consequences of its overproduction

The eukaryotic translational initiation factor 4E (eIF-4E) is an essential protein that binds the 5' cap structure with high specificity and affinity. Yeast eIF-4E is homologous to eIF-4E of higher eukaryotes, but interacts with a different set of cap-binding complex proteins. In the present study the distribution of yeast eIF-4E in Saccharomyces cerevisiae was found to be similar to that observed in higher cells, whereby the yeast factor was more concentrated in the nucleus than in the cytoplasm. Overexpression of yeast eIF-4E in S. cerevisiae exerted at most a minimal effect on growth in liquid minimal medium and was not found to influence the translation of reporter gene mRNAs bearing secondary structure in their leader regions. In a new method to study mRNA-protein interactions, biotinylated mRNAs were synthesized in vitro for use in studies of the binding of eIF-4E in yeast extracts. Streptavidin was used to adsorb the biotinylated mRNAs plus bound initiation factors. Stem-loop structures in the leader region did not influence the binding of eIF-4E or, in comparative experiments, of eIF-4A. Thus yeast eIF-4E shows both similarities and differences with respect to the distribution and function of its counterparts in higher eukaryotes.     

Phosphorylation of tobacco eukaryotic translation initiation factor 4A upon pollen tube germination

Eukaryotic translation initiation factor eIF-4A is a member of the DEAD box family of RNA helicases and RNA-dependent ATPases. In tobacco, eIF-4A is encoded by a gene family with one isoform, eIF-4A8, being exclusively expressed in pollen. This pollen-specific isoform is a candidate for mediating translational control in the developing gametophyte. Here we show that eIF-4A is barely phosphorylated in mature pollen, but during pollen tube germination, two isoforms of eIF-4A become phosphorylated. Phosphoamino acid analysis indicated phosphorylation of threonine. In order to determine whether pollen-specific eIF-4A8 is among the phosphorylated isoforms, we raised transgenic tobacco plants overexpressing eIF-4A8 containing a histidine tag. Hereby, we could show that indeed eIF-4A8 is modified through phosphorylation. The biological relevance of the phosphorylation of eIF-4A is discussed.     

Promotion of met-tRNAiMet binding to ribosomes by yIF2, a bacterial IF2 homolog in yeast

Delivery of the initiator methionine transfer RNA (Met-tRNAiMet) to the ribosome is a key step in the initiation of protein synthesis. Previous results have indicated that this step is catalyzed by the structurally dissimilar translation factors in prokaryotes and eukaryotes-initiation factor 2 (IF2) and eukaryotic initiation factor 2 (eIF2), respectively. A bacterial IF2 homolog has been identified in both eukaryotes and archaea. By using a combination of molecular genetic and biochemical studies, the Saccharomyces cerevisiae IF2 homolog is shown to function in general translation initiation by promoting Met-tRNAiMet binding to ribosomes. Thus, the mechanism of protein synthesis in eukaryotes and prokaryotes is more similar than was previously realized.     

Structure of translation initiation factor 5A from Pyrobaculum aerophilum at 1.75 A resolution

BACKGROUND: Translation initiation factor 5A (IF-5A) is reported to be involved in the first step of peptide bond formation in translation, to be involved in cell-cycle regulation and to be a cofactor for the Rev and Rex transactivator proteins of human immunodeficiency virus-1 and T-cell leukemia virus I, respectively. IF-5A contains an unusual amino acid, hypusine (N-epsilon-(4-aminobutyl-2-hydroxy)lysine), that is required for its function. The first step in the post-translational modification of lysine to hypusine is catalyzed by the enzyme deoxyhypusine synthase, the structure of which has been published recently. RESULTS: IF-5A from the archebacterium Pyrobaculum aerophilum has been heterologously expressed in Escherichia coli with selenomethionine substitution. The crystal structure of IF-5A has been determined by multiwavelength anomalous diffraction and refined to 1.75 A. Unmodified P. aerophilum IF-5A is found to be a beta structure with two domains and three separate hydrophobic cores. CONCLUSIONS: The lysine (Lys42) that is post-translationally modified by deoxyhypusine synthase is found at one end of the IF-5A molecule in an turn between beta strands beta4 and beta5; this lysine residue is freely solvent accessible. The C-terminal domain is found to be homologous to the cold-shock protein CspA of E. coli, which has a well characterized RNA-binding fold, suggesting that IF-5A is involved in RNA binding.     

Repression of cap-dependent translation by 4E-binding protein 1: competition with p220 for binding to eukaryotic initiation factor-4E

An important aspect of the regulation of gene expression is the modulation of translation rates in response to growth factors, hormones and mitogens. Most of this control is at the level of translation initiation. Recent studies have implicated the MAP kinase pathway in the regulation of translation by insulin and growth factors. MAP kinase phosphorylates a repressor of translation initiation [4E-binding protein (BP) 1] that binds to the mRNA 5' cap binding protein eukaryotic initiation factor (eIF)-4E and inhibits cap-dependent translation. Phosphorylation of the repressor decreases its affinity for eIF-4E, and thus relieves translational inhibition. eIF-4E forms a complex with two other polypeptides, eIF-4A and p220, that promote 40S ribosome binding to mRNA. Here, we have studied the mechanism by which 4E-BP1 inhibits translation. We show that 4E-BP1 inhibits 48S pre-initiation complex formation. Furthermore, we demonstrate that 4E-BP1 competes with p220 for binding to eIF-4E. Mutants of 4E-BP1 that are deficient in their binding to eIF-4E do not inhibit the interaction between p220 and eIF-4E, and do not repress translation. Thus, translational control by growth factors, insulin and mitogens is affected by changes in the relative affinities of 4E-BP1 and p220 for eIF-4E.