Archaeal translation initiation factor aIF2 can substitute for eukaryotic eIF2 in ribosomal scanning during mammalian 48S complex formation

Heterotrimeric translation initiation factor (IF) a/eIF2 (archaeal/eukaryotic IF 2) is present in both Eukarya and Archaea. Despite strong structural similarity between a/eIF2 orthologs from the two domains of life, their functional relationship is obscure. Here, we show that aIF2 from Sulfolobus solfataricus can substitute for its mammalian counterpart in the reconstitution of eukaryotic 48S initiation complexes from purified components. aIF2 is able to correctly place the initiator Met-tRNA_i into the P-site of the 40S ribosomal subunit and accompany the entire set of eukaryotic translation IFs in the process of cap-dependent scanning and AUG codon selection. However, it seems to be unable to participate in the following step of ribosomal subunit joining. In accordance with this, aIF2 inhibits rather than stimulates protein synthesis in mammalian cell-free system. The ability of recombinant aIF2 protein to direct ribosomal scanning suggests that some archaeal mRNAs may utilize this mechanism during translation initiation.     

Structure of the eukaryotic initiation factor (eIF) 5 reveals a fold common to several translation factors

Eukaryotic initiation factor 5 (eIF5) plays multiple roles in translation initiation. Its N-terminal domain functions as a GTPase-activator protein (GAP) for GTP bound to eIF2, while its C-terminal region nucleates the interactions between multiple translation factors, including eIF1, which acts to inhibit GTP hydrolysis or P(i) release, and the beta subunit of eIF2. These proteins and the events in which they participate are critical for the accurate recognition of the correct start codon during translation initiation. Here, we report the three-dimensional solution structure of the N-terminal domain of human eIF5, comprising two subdomains, both reminiscent of nucleic-acid-binding modules. The N-terminal subdomain contains the "arginine finger" motif that is essential for GAP function but which, unusually, resides in a partially disordered region of the molecule. This implies that a conformational reordering of this portion of eIF5 is likely to occur upon formation of a competent complex for GTP hydrolysis, following the appropriate activation signal. Interestingly, the N-terminal subdomain of eIF5 reveals an alpha/beta fold structurally similar to both the archaeal orthologue of the beta subunit of eIF2 and, unexpectedly, to eIF1. These results reveal a novel protein fold common to several factors involved in related steps of translation initiation. The implications of these observations are discussed in terms of the mechanism of translation initiation.     

Direct binding of translation initiation factor eIF2gamma-G domain to its GTPase-activating and GDP-GTP exchange factors eIF5 and eIF2B epsilon

The GTP-binding eukaryotic translation initiation factor eIF2 delivers initiator methionyl-tRNA to the 40 S ribosomal subunit. The factor eIF5 stimulates hydrolysis of GTP by eIF2 upon AUG codon recognition, whereas the factor eIF2B promotes guanine nucleotide exchange on eIF2 to recycle the factor for additional rounds of translation initiation. The GTP-binding (G) domain resides in the gamma subunit of the heterotrimeric eIF2; however, only eIF2beta, and not eIF2gamma, has been reported to directly bind to eIF5 or eIF2B. Using proteins expressed in yeast or recombinant systems we show that full-length yeast eIF2gamma, as well as its isolated G domain, binds directly to eIF5 and the epsilon subunit of eIF2B, and we map the interaction sites to the catalytically important regions of these factors. Consistently, an internal deletion of residues 50-100 of yeast eIF5 impairs the interaction with recombinant eIF2gamma-G domain and abolishes the ability of eIF5 to stimulate eIF2 GTPase activity in translation initiation complexes in vitro. Thus, rather than allosterically regulating eIF2gamma-G domain function via eIF2beta, our data support a model in which the GTPase-activating factor eIF5 and the guanine-nucleotide exchange factor eIF2B modulate eIF2 function through direct interactions with the eIF2gamma-G domain.     

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 regulatory beta subunit of protein kinase CK2 contributes to the recognition of the substrate consensus sequence. A study with an eIF2 beta-derived peptide

CK2 is a ubiquitous and pleiotropic Ser/Thr-specific protein kinase that phosphorylates more than 300 protein substrates at sites specified by an acidic consensus sequence in which positions n + 3 and n + 1 are particularly important. Recognition of substrates by CK2 is known to rely on basic residues located in the catalytic site of the alpha subunit which make electrostatic contacts with the negative charges in the substrate consensus sequence, thereby assuring optimal binding; the regulatory beta subunit is believed to play a protective and stabilizing role. We describe a biochemical and structural analysis of CK2-mediated phosphorylation of a 22-mer synthetic peptide corresponding to the N-terminal tail of the eukaryotic translation initiation factor eIF2beta. Results demonstrate that this peptide still displays phosphorylation features similar to full-length eIF2beta and the CK2 beta subunit also contributes to recognition of the protein substrate by establishing both polar and hydrophobic interactions with specificity determinants located downstream from the phosphoacceptor site. In particular, the N-terminal domain of the beta subunit appears to be of crucial importance for optimizing high-affinity phosphorylation of the eIF2beta peptide. This domain includes an acidic cluster whose electrostatic contacts with basic residues of the substrate attenuate intrasteric pseudosubstrate inhibition while strengthening substrate-kinase binding.     

Role of aIF5B in archaeal translation initiation

In eukaryotes and in archaea late steps of translation initiation involve the two initiation factors e/aIF5B and e/aIF1A. In eukaryotes, the role of eIF5B in ribosomal subunit joining is established and structural data showing eIF5B bound to the full ribosome were obtained. To achieve its function, eIF5B collaborates with eIF1A. However, structural data illustrating how these two factors interact on the small ribosomal subunit have long been awaited. The role of the archaeal counterparts, aIF5B and aIF1A, remains to be extensively addressed. Here, we study the late steps of Pyrococcus abyssi translation initiation. Using in vitro reconstituted initiation complexes and light scattering, we show that aIF5B bound to GTP accelerates subunit joining without the need for GTP hydrolysis. We report the crystallographic structures of aIF5B bound to GDP and GTP and analyze domain movements associated to these two nucleotide states. Finally, we present the cryo-EM structure of an initiation complex containing 30S bound to mRNA, Met-tRNA_i^Met, aIF5B and aIF1A at 2.7 Å resolution. Structural data shows how archaeal 5B and 1A factors cooperate to induce a conformation of the initiator tRNA favorable to subunit joining. Archaeal and eukaryotic features of late steps of translation initiation are discussed.     

Crystal structures of ribosome anti-association factor IF6

Ribosome anti-association factor eIF6 (originally named according to translation initiation terminology as eukaryotic initiation factor 6) binds to the large ribosomal subunit, thereby preventing inappropriate interactions with the small subunit during initiation of protein synthesis. We have determined the X-ray structures of two IF6 homologs, Methanococcus jannaschii archaeal aIF6 and Sacchromyces cerevisiae eIF6, revealing a phylogenetically conserved 25 kDa protein consisting of five quasi identical alpha/beta subdomains arrayed about a five-fold axis of pseudosymmetry. Yeast eIF6 prevents ribosomal subunit association. Comparative protein structure modeling with other known archaeal and eukaryotic homologs demonstrated the presence of two conserved surface regions, one or both of which may bind the large ribosomal subunit.     

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.     

Crystal structure of the N-terminal segment of human eukaryotic translation initiation factor 2alpha

Eukaryotic translation initiation factor 2alpha (eIF2alpha) is a member of the eIF2 heterotrimeric complex that binds and delivers Met-tRNA(i)(Met) to the 40 S ribosomal subunit in a GTP-dependent manner. Phosphorylation/dephosphorylation of eIF2alpha at Ser-51 is the major regulator of protein synthesis in eukaryotic cells. Here, we report the first structural analysis on eIF2, the three-dimensional structure of a 22-kDa N-terminal portion of human eIF2alpha by x-ray diffraction at 1.9 A resolution. This structure contains two major domains. The N terminus is a beta-barrel with five antiparallel beta-strands in an oligonucleotide binding domain (OB domain) fold. The phosphorylation site (Ser-51) is on the loop connecting beta3 and beta4 in the OB domain. A helical domain follows the OB domain, and the first helix has extensive interactions, including a disulfide bridge, to fix its orientation with respect to the OB domain. The two domains meet along a negatively charged groove with highly conserved residues, indicating a likely site for protein-protein interaction.     

Structure-function relationships of the intact aIF2alpha subunit from the archaeon Pyrococcus abyssi

Eukaryotic and archaeal initiation factor 2 (e- and aIF2, respectively) are heterotrimeric proteins (alphabetagamma) supplying the small subunit of the ribosome with methionylated initiator tRNA. The gamma subunit forms the core of the heterotrimer. It resembles elongation factor EF1-A and ensures interaction with Met-tRNA(i)(Met). In the presence of the alpha subunit, which is composed of three domains, the gamma subunit expresses full tRNA binding capacity. This study reports the crystallographic structure of the intact aIF2alpha subunit from the archaeon Pyrococcus abyssi and that of a derived C-terminal fragment containing domains 2 and 3. The obtained structures are compared with those of N-terminal domains 1 and 2 of yeast and human eIF2alpha and with the recently determined NMR structure of human eIF2alpha. We show that the three-domain organization in the alpha subunit is conserved in archaea and eukarya. Domains 1 and 2 form a rigid body linked to a mobile third domain. Sequence comparisons establish that the most conserved regions in the aIF2alpha polypeptide lie at opposite sides of the protein, within domain 1 and domain 3, respectively. These two domains are known to exhibit RNA binding capacities. We propose that domain 3, which is known to glue the alpha subunit onto the gamma subunit, participates in Met-tRNA(i)(Met) binding while domain 1 recognizes either rRNA or mRNA on the ribosome. Thereby, the observed structural mobility within the e- and aIF2alpha molecules would be an integral part of the biological function of this subunit in the heterotrimeric e- and aIF2alphabetagamma factors.     

Structure and interactions of the translation initiation factor eIF1

eIF1 is a universally conserved translation factor that is necessary for scanning and involved in initiation site selection. We have determined the solution structure of human eIF1 with an N-terminal His tag using NMR spectroscopy. Residues 29-113 of the native sequence form a tightly packed domain with two alpha-helices on one side of a five-stranded parallel and antiparallel beta-sheet. The fold is new but similar to that of several ribosomal proteins and RNA-binding domains. A likely binding site is indicated by yeast mutations and conserved residues located together on the surface. No interaction with recombinant eIF5 or the initiation site RNA GCCACAAUGGCA was detected by NMR, but GST pull-down experiments show that eIF1 binds specifically to the p110 subunit of eIF3. This interaction explains how eIF1 is recruited to the 40S ribosomal subunit.     

In vitro phosphorylation of initiation factor 2 alpha (aIF2 alpha) from hyperthermophilic archaeon Pyrococcus horikoshii OT3

Eukaryotic initiation factor 2 (eIF2) is a heterotrimeric protein composed of alpha, beta, and gamma subunits, of which the alpha subunit (eIF2 alpha) plays a crucial role in regulation of protein synthesis through phosphorylation at Ser51. All three subunit genes are conserved in Archaea. To examine the properties of archaeal initiation factor 2 alpha (aIF2 alpha), three genes encoding alpha, beta, and gamma subunits of aIF2 from the hyperthermophilic archaeon Pyrococcus horikoshii OT3 were expressed in Escherichia coli cells, and the resulting proteins, aIF2 alpha, aIF2 beta, and aIF2 gamma, were characterized with reference to the properties of eIF2. aIF2 alpha preferentially interacts with aIF2 gamma, but does not interact with aIF2 beta, which is consistent with data obtained with eIF2, of which eIF2 gamma serves as a core subunit, interacting with eIF2 alpha and eIF2 beta. It was found that aIF2 alpha was, albeit to a lower degree, phosphorylated by double-stranded RNA-dependent protein kinase (hPKR) from human, and a primary target site was suggested to be Ser48 within aIF2 alpha. This finding led us to the search for a putative aIF2 specific kinase gene (PH0512) in the P. horikoshii genome. The gene product Ph0512p unambiguously phosphorylated aIF2 alpha, and Ser48, as in the phosphorylation by hPKR, was suggested to be a target amino acid residue for the PKR homologue Ph0152p in P. horikoshii. These findings suggest that aIF2 alpha, like eIF2 alpha in eukaryotes, plays a role in regulation of the protein synthesis in Archaea through phosphorylation and dephosphorylation.     

Archaeal aIF2B Interacts with Eukaryotic Translation Initiation Factors eIF2α and eIF2Bα: Implications for aIF2B Function and eIF2B Regulation

Translation initiation is down-regulated in eukaryotes by phosphorylation of the α-subunit of eIF2 (eukaryotic initiation factor 2), which inhibits its guanine nucleotide exchange factor, eIF2B. The N-terminal S1 domain of phosphorylated eIF2α interacts with a subcomplex of eIF2B formed by the three regulatory subunits α/GCN3, β/GCD7, and δ/GCD2, blocking the GDP-GTP exchange activity of the catalytic ?-subunit of eIF2B. These regulatory subunits have related sequences and have sequences in common with many archaeal proteins, some of which are involved in methionine salvage and CO₂ fixation. Our sequence analyses however predicted that members of one phylogenetically distinct and coherent group of these archaeal proteins [designated aIF2Bs (archaeal initiation factor 2Bs)] are functional homologs of the α, β, and δ subunits of eIF2B. Three of these proteins, from different archaea, have been shown to bind in vitro to the α-subunit of the archaeal aIF2 from the cognate archaeon. In one case, the aIF2B protein was shown further to bind to the S1 domain of the α-subunit of yeast eIF2 in vitro and to interact with eIF2Bα/GCN3 in vivo in yeast. The aIF2B-eIF2α interaction was however independent of eIF2α phosphorylation. Mass spectrometry has identified several proteins that co-purify with aIF2B from Thermococcus kodakaraensis, and these include aIF2α, a sugar-phosphate nucleotidyltransferase with sequence similarity to eIF2B?, and several large-subunit (50S) ribosomal proteins. Based on this evidence that aIF2B has functions in common with eIF2B, the crystal structure established for an aIF2B was used to construct a model of the eIF2B regulatory subcomplex. In this model, the evolutionarily conserved regions and sites of regulatory mutations in the three eIF2B subunits in yeast are juxtaposed in one continuous binding surface for phosphorylated eIF2α.     

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.     

X-ray structure of translation initiation factor eIF2gamma: implications for tRNA and eIF2alpha binding

The x-ray structure of the gamma-subunit of the heterotrimeric translation initiation factor eIF2 has been determined to 2.4-A resolution. eIF2 is a GTPase that delivers the initiator Met-tRNA to the P site on the small ribosomal subunit during a rate-limiting initiation step in translation. The structure of eIF2gamma closely resembles that of EF1A.GTP, consisting of an N-terminal G domain followed by two beta-barrels arranged in a closed configuration with domain II packed against the G domain in the vicinity of the Switch regions. The G domain of eIF2gamma has an unusual zinc ribbon motif, not previously found in other GTPases. Structure-based site-directed mutagenesis was used to identify two adjacent features on the surface of eIF2gamma that bind the alpha-subunit and Met-tRNA(i)(Met), respectively. These structural, biochemical, and genetic results provide new insights into eIF2 ternary complex assembly.     

Conserved bipartite motifs in yeast eIF5 and eIF2Bepsilon, GTPase-activating and GDP-GTP exchange factors in translation initiation, mediate binding to their common substrate eIF2

In the initiation phase of eukaryotic translation, eIF5 stimulates the hydrolysis of GTP bound to eIF2 in the 40S ribosomal pre-initiation complex, and the resultant GDP on eIF2 is replaced with GTP by the complex nucleotide exchange factor, eIF2B. Bipartite motifs rich in aromatic and acidic residues are conserved at the C-termini of eIF5 and the catalytic (epsilon) subunit of eIF2B. Here we show that these bipartite motifs are important for the binding of these factors, both in vitro and in vivo, to the beta subunit of their common substrate eIF2. We also find that three lysine-rich boxes in the N-terminal segment of eIF2beta mediate the binding of eIF2 to both eIF5 and eIF2B. Thus, eIF5 and eIF2Bepsilon employ the same sequence motif to facilitate interaction with the same segment of their common substrate. In agreement with this, archaea appear to lack eIF5, eIF2B and the lysine-rich binding domain for these factors in their eIF2beta homolog. The eIF5 bipartite motif is also important for its interaction with the eIF3 complex through the NIP1-encoded subunit of eIF3. Thus, the bipartite motif in eIF5 appears to be multifunctional, stimulating its recruitment to the 40S pre-initiation complex through interaction with eIF3 in addition to binding of its substrate eIF2.     

The large subunit of initiation factor aIF2 is a close structural homologue of elongation factors

The heterotrimeric factor e/aIF2 plays a central role in eukaryotic/archaeal initiation of translation. By delivering the initiator methionyl-tRNA to the ribosome, e/aIF2 ensures specificity of initiation codon selection. The three subunits of aIF2 from the hyperthermophilic archaeon Pyrococcus abyssi could be overproduced in Escherichia coli. The beta and gamma subunits each contain a tightly bound zinc. The large gamma subunit is shown to form the structural core for trimer assembly. The crystal structures of aIF2gamma, free or complexed to GDP-Mg(2+) or GDPNP-Mg(2+), were resolved at resolutions better than 2 A. aIF2gamma displays marked similarities to elongation factors. A distinctive feature of e/aIF2gamma is a subdomain containing a zinc-binding knuckle. Examination of the nucleotide-complexed aIF2gamma structures suggests mechanisms of action and tRNA binding properties similar to those of an elongation factor. Implications for the mechanism of translation initiation in both eukarya and archaea are discussed. In particular, positioning of the initiator tRNA in the ribosomal A site during the search for the initiation codon is envisaged.     

PKR and GCN2 kinases and guanine nucleotide exchange factor eukaryotic translation initiation factor 2B (eIF2B) recognize overlapping surfaces on eIF2alpha

Four stress-responsive protein kinases, including GCN2 and PKR, phosphorylate eukaryotic translation initiation factor 2alpha (eIF2alpha) on Ser51 to regulate general and gene-specific protein synthesis. Phosphorylated eIF2 is an inhibitor of its guanine nucleotide exchange factor, eIF2B. Mutations that block translational regulation were isolated throughout the N-terminal OB-fold domain in Saccharomyces cerevisiae eIF2alpha, including those at residues flanking Ser51 and around 20 A away in the conserved motif K79GYID83. Any mutation at Glu49 or Asp83 blocked translational regulation; however, only a subset of these mutations impaired Ser51 phosphorylation. Substitution of Ala for Asp83 eliminated phosphorylation by GCN2 and PKR both in vivo and in vitro, establishing the critical contributions of remote residues to kinase-substrate recognition. In contrast, mutations that blocked translational regulation but not Ser51 phosphorylation impaired the binding of eIF2B to phosphorylated eIF2alpha. Thus, two structurally distinct effectors of eIF2 function, eIF2alpha kinases and eIF2B, have evolved to recognize the same surface and overlapping determinants on eIF2alpha.     

Solution structure of human initiation factor eIF2alpha reveals homology to the elongation factor eEF1B

The GTP-bound form of the trimeric eukaryotic translation initiation factor 2 (eIF2) transfers aminoacylated initiator methionyl tRNA onto the 40S ribosome. We have solved with solution NMR the structure of the alpha subunit of human eIF2 (heIF2alpha). The protein consists of two domains that are mobile relative to each other. The N-terminal domain has an S1-type oligonucleotide/oligosaccharide binding-fold subdomain and an alpha-helical subdomain. The C-terminal domain adopts an alphabeta-fold very similar to the C-terminal domain of elongation factor (eEF) 1Balpha, the guanine-nucleotide exchange factor for eEF1A. The structural and functional similarities found between eIF2alpha/eIF2gamma and eEF1Balpha/eEF1A suggest a model for the interaction of eIF2alpha with eIF2gamma, and eIF2 with Met-tRNAiMet. It further indicates a previously unrecognized evolutionary lineage of eIF2alpha/gamma from the functionally related elongation factor eEF1Balpha/eEF1A complex.     

Mutational analysis of mammalian translation initiation factor 5 (eIF5): role of interaction between the beta subunit of eIF2 and eIF5 in eIF5 function in vitro and in vivo

Eukaryotic translation initiation factor 5 (eIF5) interacts with the 40S initiation complex (40S-eIF3-AUG-Met-tRNA(f)-eIF2-GTP) to promote the hydrolysis of ribosome-bound GTP. eIF5 also forms a complex with eIF2 by interacting with the beta subunit of eIF2. In this work, we have used a mutational approach to investigate the importance of eIF5-eIF2beta interaction in eIF5 function. Binding analyses with recombinant rat eIF5 deletion mutants identified the C terminus of eIF5 as the eIF2beta-binding region. Alanine substitution mutagenesis at sites within this region defined several conserved glutamic acid residues in a bipartite motif as critical for eIF5 function. The E346A,E347A and E384A,E385A double-point mutations each caused a severe defect in the binding of eIF5 to eIF2beta but not to eIF3-Nip1p, while a eIF5 hexamutant (E345A,E346A, E347A,E384A,E385A,E386A) showed negligible binding to eIF2beta. These mutants were also severely defective in eIF5-dependent GTP hydrolysis, in 80S initiation complex formation, and in the ability to stimulate translation of mRNAs in an eIF5-dependent yeast cell-free translation system. Furthermore, unlike wild-type rat eIF5, which can functionally substitute for yeast eIF5 in complementing in vivo a genetic disruption of the chromosomal copy of the TIF5 gene, the eIF5 double-point mutants allowed only slow growth of this DeltaTIF5 yeast strain, while the eIF5 hexamutant was unable to support cell growth and viability of this strain. These findings suggest that eIF5-eIF2beta interaction plays an essential role in eIF5 function in eukaryotic cells.