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.     

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.     

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.     

The phosphorylation state of translation initiation factors is regulated developmentally and following heat shock in wheat

Several translation initiation factors in mammals and yeast are regulated by phosphorylation. The phosphorylation state of these factors is subject to alteration during development, environmental stress (heat shock, starvation, or heme deprivation), or viral infection. The phosphorylation state and the effect of changes in phosphorylation of the translation initiation factors of higher plants have not been previously investigated. We have determined the isoelectric states for the wheat translation initiation factors eIF-4A, eIF-4B, eIF-4F, eIF-iso4F, and eIF-2 and the poly(A)-binding protein in the seed, during germination, and following heat shock of wheat seedlings using two-dimensional gel electrophoresis and Western analysis. We found that the developmentally induced changes in isoelectric state observed during germination or the stress-induced changes were consistent with changes in phosphorylation. Treatment of the phosphorylated forms of the factors with phosphatases confirmed that the nature of the modification was due to phosphorylation. The isoelectric states of eIF-4B, eIF-4F (eIF-4E, p26), eIF-iso4F (eIF-iso4E, p28), and eIF-2alpha (p42) were altered during germination, suggesting that phosphorylation of these factors is developmentally regulated and correlates with the resumption of protein synthesis that occurs during germination. The phosphorylation of eIF-2beta (p38) or poly(A)-binding protein did not change either during germination or following a thermal stress. Only the phosphorylation state of two factors, eIF-4A and eIF-4B, changed following a heat shock, suggesting that plants may differ significantly from animals in the way in which their translational machinery is modified in response to a thermal stress.     

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.     

Archaeal translation initiation revisited: the initiation factor 2 and eukaryotic initiation factor 2B alpha-beta-delta subunit families

As the amount of available sequence data increases, it becomes apparent that our understanding of translation initiation is far from comprehensive and that prior conclusions concerning the origin of the process are wrong. Contrary to earlier conclusions, key elements of translation initiation originated at the Universal Ancestor stage, for homologous counterparts exist in all three primary taxa. Herein, we explore the evolutionary relationships among the components of bacterial initiation factor 2 (IF-2) and eukaryotic IF-2 (eIF-2)/eIF-2B, i.e., the initiation factors involved in introducing the initiator tRNA into the translation mechanism and performing the first step in the peptide chain elongation cycle. All Archaea appear to posses a fully functional eIF-2 molecule, but they lack the associated GTP recycling function, eIF-2B (a five-subunit molecule). Yet, the Archaea do posses members of the gene family defined by the (related) eIF-2B subunits alpha, beta, and delta, although these are not specifically related to any of the three eukaryotic subunits. Additional members of this family also occur in some (but by no means all) Bacteria and even in some eukaryotes. The functional significance of the other members of this family is unclear and requires experimental resolution. Similarly, the occurrence of bacterial IF-2-like molecules in all Archaea and in some eukaryotes further complicates the picture of translation initiation. Overall, these data lend further support to the suggestion that the rudiments of translation initiation were present at the Universal Ancestor stage.     

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.     

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.     

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.     

Disruption of cellular translational control by a viral truncated eukaryotic translation initiation factor 2alpha kinase homolog

Phosphorylation of eukaryotic translation initiation factor 2alpha (eIF2alpha) is a common cellular mechanism to limit protein synthesis in stress conditions. Baculovirus PK2, which resembles the C-terminal half of a protein kinase domain, was found to inhibit both human and yeast eIF2alpha kinases. Insect cells infected with wild-type, but not pk2-deleted, baculovirus exhibited reduced eIF2alpha phosphorylation and increased translational activity. The negative regulatory effect of human protein kinase RNA-regulated (PKR), an eIF2alpha kinase, on virus production was counteracted by PK2, indicating that baculoviruses have evolved a unique strategy for disrupting a host stress response. PK2 was found in complex with PKR and blocked kinase autophosphorylation in vivo, suggesting a mechanism of kinase inhibition mediated by interaction between truncated and intact kinase domains.     

Using GCN4 as a reporter of eIF2 alpha phosphorylation and translational regulation in yeast

Molecular genetic analyses in yeast are a powerful method to study gene regulation. Conservation of the mechanism and regulation of protein synthesis between yeast and mammalian cells makes yeast a good model system for the analysis of translation. One of the most common mechanisms of translational regulation in mammalian cells is the phosphorylation of serine-51 on the alpha subunit of the translation initiation factor elF2, which causes an inhibition of general translation. In contrast, in the yeast Saccharomyces cerevisiae phosphorylation of elF2 alpha on serine-51 by the GCN2 protein kinase mediates the translational induction of GCN4 expression. The unique structure of the GCN4 mRNA makes GCN4 expression especially sensitive to elF2 alpha phosphorylation, and the simple microbiological tests developed in yeast to analyze GCN4 expression serve as good reporters of elF2 alpha phosphorylation. It is relatively simple to express heterologous proteins in yeast, and it has been shown that the mammalian elF2 alpha kinases will functionally substitute for GCN2. Structure-function analyses of translation factors or translational regulators can also be performed by assaying for effects on general and GCN4-specific translation. Three tests can be used to study elF2 alpha phosphorylation and/or translational activity in yeast. First, general translation can be monitored by simple growth tests, while GCN4 expression can be analyzed using sensitive replicaplating tests. Second, GCN4 translation can be quantitated by measuring expression from GCN4-lacZ reporter constructs. Finally, isoelectric focusing gels can be used to directly monitor in vivo phosphorylation of elF2 alpha in yeast.     

A ribosomal protein is required for translational regulation of GCN4 mRNA. Evidence for involvement of the ribosome in eIF2 recycling

In amino acid-starved yeast cells, inhibition of the guanine nucleotide exchange factor eIF2B by phosphorylated translation initiation factor 2 results in increased translation of GCN4 mRNA. We isolated a suppressor of a mutant eIF2B. The suppressor prevents efficient GCN4 mRNA translation due to inactivation of the small ribosomal subunit protein Rps31 and results in low amounts of mutant 40 S ribosomal subunits. Deletion of one of two genes encoding ribosomal protein Rps17 also reduces the amounts of 40 S subunits but does not suppress eIF2B mutations or prevent efficient GCN4 translation. Our findings show that Rps31-deficient ribosomes are altered in a way that decreases the eIF2B requirement and that the small ribosomal subunit mediates the effects of low eIF2B activity on cell viability and translational regulation in response to eIF2 phosphorylation.     

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.     

Homologous segments in three subunits of the guanine nucleotide exchange factor eIF2B mediate translational regulation by phosphorylation of eIF2

eIF2B is a five-subunit guanine nucleotide exchange factor that is negatively regulated by phosphorylation of the alpha subunit of its substrate, eIF2, leading to inhibition of translation initiation. To analyze this regulatory mechanism, we have characterized 29 novel mutations in the homologous eIF2B subunits encoded by GCD2, GCD7, and GCN3 that reduce or abolish inhibition of eIF2B activity by eIF2 phosphorylated on its alpha subunit [eIF2(alphaP)]. Most, if not all, of the mutations decrease sensitivity to eIF2(alphaP) without excluding GCN3, the nonessential subunit, from eIF2B; thus, all three proteins are critical for regulation of eIF2B by eIF2(alphaP). The mutations are clustered at both ends of the homologous region of each subunit, within two segments each of approximately 70 amino acids in length. Several mutations alter residues at equivalent positions in two or all three subunits. These results imply that structurally similar segments in GCD2, GCD7, and GCN3 perform related functions in eIF2B regulation. We propose that these segments form a single domain in eIF2B that makes multiple contacts with the alpha subunit of eIF2, around the phosphorylation site, allowing eIF2B to detect and respond to phosphoserine at residue 51. Most of the eIF2 is phosphorylated in certain mutants, suggesting that these substitutions allow eIF2B to accept phosphorylated eIF2 as a substrate for nucleotide exchange.     

The herpes simplex virus US11 protein effectively compensates for the gamma1(34.5) gene if present before activation of protein kinase R by precluding its phosphorylation and that of the alpha subunit of eukaryotic translation initiation factor 2

In herpes simplex virus-infected cells, viral gamma134.5 protein blocks the shutoff of protein synthesis by activated protein kinase R (PKR) by directing the protein phosphatase 1alpha to dephosphorylate the alpha subunit of eukaryotic translation initiation factor 2 (eIF-2alpha). The amino acid sequence of the gamma134.5 protein which interacts with the phosphatase has high homology to a domain of the eukaryotic protein GADD34. A class of compensatory mutants characterized by a deletion which results in the juxtaposition of the alpha47 promoter next to US11, a gamma2 (late) gene in wild-type virus-infected cells, has been described. In cells infected with these mutants, protein synthesis continues even in the absence of the gamma134.5 gene. In these cells, PKR is activated but eIF-2alpha is not phosphorylated, and the phosphatase is not redirected to dephosphorylate eIF-2alpha. We report the following: (i) in cells infected with these mutants, US11 protein was made early in infection; (ii) US11 protein bound PKR and was phosphorylated; (iii) in in vitro assays, US11 blocked the phosphorylation of eIF-2alpha by PKR activated by poly(I-C); and (iv) US11 was more effective if present in the reaction mixture during the activation of PKR than if added after PKR had been activated by poly(I-C). We conclude the following: (i) in cells infected with the compensatory mutants, US11 made early in infection binds to PKR and precludes the phosphorylation of eIF-2alpha, whereas US11 driven by its natural promoter and expressed late in infection is ineffective; and (ii) activation of PKR by double-stranded RNA is a common impediment countered by most viruses by different mechanisms. The gamma134.5 gene is not highly conserved among herpesviruses. A likely scenario is that acquisition by a progenitor of herpes simplex virus of a portion of the cellular GADD34 gene resulted in a more potent and reliable means of curbing the effects of activated PKR. US11 was retained as a gamma2 gene because, like many viral proteins, it has multiple functions.     

Ribosome targeting of PKR is mediated by two double-stranded RNA-binding domains and facilitates in vivo phosphorylation of eukaryotic initiation factor-2

Protein kinase PKR is activated in mammalian cells during viral infection, leading to phosphorylation of the alpha subunit of eukaryotic initiation factor-2 (eIF-2alpha) and inhibition of protein synthesis. This antiviral response is thought to be mediated by association of double-stranded RNA (ds-RNA), a by-product of viral replication, with two ds-RNA-binding domains (DRBDs) located in the amino terminus of PKR. Recent studies have observed that expression of mammalian PKR in yeast leads to a slow growth phenotype due to hyperphosphorylation of eIF-2alpha. In this report, we observed that while DRBD sequences are required for PKR to function in the yeast model system, these sequences are not required for in vitro phosphorylation of eIF-2alpha. To explain this apparent contradiction, we proposed that these sequences are required to target the kinase to the translation machinery. Using sucrose gradient sedimentation, we found that wild-type PKR was associated with ribosomes, specifically with 40 S particles. Deletions or residue substitutions in the DRBD sequences blocked kinase interaction with ribosomes. These results indicate that in addition to mediating ds-RNA control of PKR, the DRBD sequences facilitate PKR association with ribosomes. Targeting to ribosomes may enhance in vivo phosphorylation of eIF-2alpha, by providing PKR access to its substrate.     

GTP hydrolysis controls stringent selection of the AUG start codon during translation initiation in Saccharomyces cerevisiae

We have isolated and characterized two suppressor genes, SUI4 and SUI5, that can initiate translation in the absence of an AUG start codon at the HIS4 locus in Saccharomyces cerevisiae. Both suppressor genes are dominant in diploid cells and lethal in haploid cells. The SUI4 suppressor gene is identical to the GCD11 gene, which encodes the gamma subunit of the eIF-2 complex and contains a mutation in the G2 motif, one of the four signature motifs that characterizes this subunit to be a G-protein. The SUI5 suppressor gene is identical to the TIF5 gene that encodes eIF-5, a translation initiation factor known to stimulate the hydrolysis of GTP bound to eIF-2 as part of the 43S preinitiation complex. Purified mutant eIF-5 is more active in stimulating GTP hydrolysis in vitro than wild-type eIF-5, suggesting that an alteration of the hydrolysis rate of GTP bound to the 43S preinitiation complex during ribosomal scanning allows translation initiation at a non-AUG codon. Purified mutant eIF-2gamma complex is defective in ternary complex formation and this defect correlates with a higher rate of dissociation from charged initiator-tRNA in the absence of GTP hydrolysis. Biochemical characterization of SUI3 suppressor alleles that encode mutant forms of the beta subunit of eIF-2 revealed that these mutant eIF-2 complexes have a higher intrinsic rate of GTP hydrolysis, which is eIF-5 independent. All of these biochemical defects result in initiation at a UUG codon at the his4 gene in yeast. These studies in light of other analyses indicate that GTP hydrolysis that leads to dissociation of eIF-2 x GDP from the initiator-tRNA in the 43S preinitiation complex serves as a checkpoint for a 3-bp codon/anticodon interaction between the AUG start codon and the initiator-tRNA during the ribosomal scanning process.     

Two structural domains of initiation factor eIF-4B are involved in binding to RNA

Translation initiation factor eIF-4B promotes the binding of mRNA to 40 S preinitiation complexes and together with eIF-4A possesses RNA helicase activity. To elucidate structural features involved in its function, a series of internal and C-terminal deletions, as well as point mutations, were constructed in the eIF-4B cDNA. The mutated cDNAs were expressed in transiently transfected COS-1 cells, and mutant forms of the factor were overproduced up to about 25-fold over endogenous eIF-4B levels. Inhibition of dihydrofolate reductase (DHFR) synthesis by high levels of eIF-4B variants was determined in vivo, and the binding of the eIF-4B forms to biotinylated RNA was measured in vitro. The results indicate that the N-terminal region containing the RNA binding motif with its RNP1 and RNP2 consensus elements is sufficient for inhibition of DHFR synthesis. Deletion of the RNP1 sequence abrogates RNA binding, but amino acid substitutions at conserved residues do not always inhibit RNA binding. Deletion of the DRYG domain near the middle of eIF-4B results in inhibition of RNA binding, but not of DHFR synthesis. Up to 164 residues of the C terminus are not required for RNA binding, but removal of 226 or more residues completely inhibits RNA binding, perhaps by the loss of two arginine-rich regions. The results suggest that both the RNA recognition motif and the arginine-rich region are required for stable RNA binding but that both are not necessary for in vivo inhibition of protein synthesis.