Eukaryotic initiation factor eIF2

eIF2 plays a central role in the maintenance of what is generally considered a rate-limiting step in mRNA translation. In this step, eIF2 binds GTP and Met-tRNAi and transfers Met-tRNAi to the 40S ribosomal subunit. At the end of the initiation process, GTP bound to eIF2 is hydrolyzed to GDP and the eIF2.GDP complex is released from the ribosome. The exchange of GDP bound to eIF2 for GTP is a prerequisite to binding Met-tRNAi and is mediated by a second initiation factor, eIF2B. In what is probably the best-characterized mechanism for the regulation of mRNA translation, phosphorylation of eIF2 on its smallest, or alpha-, subunit converts eIF2 from a substrate of eIF2B into a competitive inhibitor. Thus, phosphorylation of eIF2 alpha effectively prevents formation of the eIF2.GTP.Met-tRNAi complex and inhibits global protein synthesis. Phosphorylation of eIF2 alpha occurs under a variety of conditions including viral infection, apoptosis, nutrient deprivation, heme-deprivation, and certain stresses.     

Identification and initial characterization of translational initiation factor 2 from bovine mitochondria

The bovine liver mitochondrial factor that promotes the binding of fMet-tRNA to mitochondrial ribosomes, initiation factor 2 (IF-2mt), has been identified in the postribosomal supernatant fraction of isolated liver mitochondria. This factor has been purified approximately 5,000-fold and present preparations are estimated to be about 10% pure. IF-2mt has an apparent molecular weight of about 140,000 as determined by gel filtration chromatography. IF-2mt is active in stimulating fMet-tRNA binding to Escherichia coli ribosomes but E. coli IF-2 is not active in promoting initiator tRNA binding to animal mitochondrial ribosomes. The IF-2mt-mediated binding of fMet-tRNAi(Met) to mitochondrial ribosomes is dependent on the presence of a message such as poly(A,U,G) and on GTP. Nonhydrolyzable analogs of GTP are 2-3-fold less effective in promoting initiation complex formation on mitochondrial ribosomes than is GTP suggesting that IF-2mt is capable of recycling to some extent under the current assay conditions.     

Purification and properties of a methionine formylmethionyl-tRNA-binding initiation factor from pig liver

(i) A factor, EIF-2, that binds methionyl-tRNA_f^Met in the presence of GTP has been isolated from pig liver. (ii) Dodecylsulfate-gel electrophoresis and sedimentation equilibrium centrifugation indicate that the factor has a molecular weight of 122,000 and that it consists of three unequal subunits. (iii) The apparent K_D for binding of methionyl-tRNA_f^Met varies with factor concentration. GTP participates in the binding with a K_D of 0.5 μm. β,γ-Methylene-guanosine triphosphate supports 40% of the binding observed with GTP. GDP is a competitive inhibitor with a K_i of 0.2 μm. The optimal, free Mg²⁺ concentration is approximately 50 μm. GTP and Mg²⁺ stabilize the factor against thermal inactivation and inactivation by N-ethyl maleimide. (iv) The factor is required for the formation of a sucrose gradient-stable complex between methionyl-tRNA_f^Met and the 40S ribosomal subunit. The presence of template is not necessary, but poly(A,U,G) increases the binding observed 1.5-fold. (v) The factor markedly stimulates synthesis in a reconstituted protein-synthesizing system with globin messenger RNA as template.     

Function of initiation factor 1 in the binding and release of initiation factor 2 from ribosomal initiation complexes in Escherichia coli.

1. Studies on the function of initiation factor 1 (IF-1) in the formation of 30 S initiation complexes have been carried out. IF-1 appears to prevent the dissociation of initiation factor 2 (IF-2) from the 30 S initiation complex. The factor has no effect on either the initial binding of IF-2 nor does it increase the amount of IF-2 dependent fMet-tRNA and GTP bound to the 30 S subunit. Bound fMet-tRNA remains stable to sucrose gradient centrifugation even in the absence of IF-1. 2. It is postulated that the presence of IF-2 on the 30 S complex is necessary so that at the time of junction with the 50 S subunit to form a 70 S complex, the 70 S-dependent GTPase activity of IF-2 can hydrolyze GTP. This hydrolysis provides a means by which GTP can be removed to facilitate formation of a 70 S initiation complex active in peptidyl transfer. In support of this postulate, it was observed that 30 S initiation complexes formed in the absence of IF-1 could be depleted of their complexes were still able to accept 50 S subunits to form 70 S complexes which could still donate fMet-tRNA into peptide linkages. These results indicate that 30 S complexes lacking GTP do not require IF-2 for formation of active 70 S complexes. 3. IF-1, which is required to prevent dissociation of IF-2 from the 30 S initiation complex, is also required for release of IF-2 from ribosomes following 70 S initiation complex formation. The mechanisms of the release of IF-2 has been studied in greater detail. Evidence is presented which rules out the presence of a stable IF-2 GDP complex on the surface of the 70 S ribosome following GTP hydrolysis and of any exchange reactions between IF-1 and guanine nucleotides necessary for effecting the release of IF-2. IF-2 remains on the 70 S initiation complexes after release of guanine nucleotides and can be liberated solely by addition of IF-1.     

The involvement of a complex between formylmethionyl-tRNA and initiation factor IF-2 in prokaryotic initiation.

A complex between initiation factor IF-2 and fMet-tRNA can be formed under ionic conditions, which are optimal for initiation complex formation. The complex can be retained on cellulose nitrate filters after fixing with glutaraldehyde. The IF-2 - FMet-tRNA complex formation is not influenced by GTP and GDP. Other nucleoside di of triphosphates also have no effect. Evidence is presented that this complex acts as an intermediate in polypeptide chain initiation. The IF-2 - fMet-tRNA complex formation is not influenced by initiation factors IF-1 and IF-3. The binary complex can be bound to the 30-S subunit in the absence of GTP, which indicates that there is no concomittant binding of the IF-2 - fMet-tRNA complex and the nucleotide moiety to the 30-S subunit. The binding of the binary complex is stimulated by GTP. The influence of some inhibitors of initiation on the IF-2 - fMet-tRNA complex formation has been tested. Aurin tricarboxylic acid appeared to be a strong inhibitor, whereas the sulfhydryl reagents N-ethylmaleimide and p-chloromercuribenzoate had no effect.     

Identification of a second GTP-bound magnesium ion in archaeal initiation factor 2

Eukaryotic and archaeal translation initiation processes involve a heterotrimeric GTPase e/aIF2 crucial for accuracy of start codon selection. In eukaryotes, the GTPase activity of eIF2 is assisted by a GTPase-activating protein (GAP), eIF5. In archaea, orthologs of eIF5 are not found and aIF2 GTPase activity is thought to be non-assisted. However, no in vitro GTPase activity of the archaeal factor has been reported to date. Here, we show that aIF2 significantly hydrolyses GTP in vitro. Within aIF2γ, H97, corresponding to the catalytic histidine found in other translational GTPases, and D19, from the GKT loop, both participate in this activity. Several high-resolution crystal structures were determined to get insight into GTP hydrolysis by aIF2γ. In particular, a crystal structure of the H97A mutant was obtained in the presence of non-hydrolyzed GTP. This structure reveals the presence of a second magnesium ion bound to GTP and D19. Quantum chemical/molecular mechanical simulations support the idea that the second magnesium ion may assist GTP hydrolysis by helping to neutralize the developing negative charge in the transition state. These results are discussed in light of the absence of an identified GAP in archaea to assist GTP hydrolysis on aIF2.     

Partial reaction of peptide initiation inhibited by the reticulocyte hemin-controlled repressor

The hemin-controlled repressor from rabbit reticulocytes inhibits binding of Met-tRNA_f to reticulocyte 40S ribosomal subunits in a partial reaction containing these components, two initiation factor fractions and GTP. The inhibitor does not interfere with the formation of the Met-tRNA_f· initiation factor IF-E₂·GTP complex.     

Guanine nucleotides in protein synthesis. Utilization of pppGpp and dGTP by initiation factor 2 and elongation factor Tu

Guanosine 5′-triphosphate, 3′-diphosphate (pppGpp), and dGTP support the initiation factor 2 (IF-2) and elongation factor Tu (EF-Tu) partial reactions of Escherichia coli protein synthesis. These natural analogs of GTP were as effective as GTP in supporting (1) IF-2-dependent binding of f-Met-tRNA to ribosomes, (2) IF-2-dependent formation of N-formylmethionylpuromycin, (3) EF-Tu-dependent binding of Phe-tRNA to a ribosome-polyuridylic acid-N-acetyl-Phe-tRNA complex, and (4) EF-Tu-dependent formation of N-acetyl-Phe-Phe-tRNA. GTP, pppGpp, and dGTP behaved similarly in time-course studies and across a broad concentration range with both enzymes. In addition, both GDP and guanosine 5′-diphosphate, 3′-diphosphate were found to be competitive inhibitors of both GTP and pppGpp in the IF-2- and EF-Tu-dependent reactions.     

Activation of cap-independent translation by variant eukaryotic initiation factor 4G in vivo

Protein synthesis is tightly controlled by assembly of an intricate ribonucleoprotein complex at the m⁷GTP-cap on eukaryotic mRNAs. Ensuing linear scanning of the 5′ untranslated region (UTR) is believed to transfer the preinitiation complex to the initiation codon. Eukaryotic mRNAs are characterized by significant 5′ UTR heterogeneity, raising the possibility of differential control of translation initiation rate at individual mRNAs. Curiously, many mRNAs with unconventional, highly structured 5′ UTRs encode proteins with central biological roles in growth control, metabolism, or stress response. The 5′ UTRs of such mRNAs may influence protein synthesis rate in multiple ways, but most significantly they have been implicated in mediating alternative means of translation initiation. Cap-independent initiation bypasses strict control over the formation of initiation intermediates at the m⁷GTP cap. However, the molecular mechanisms that favor alternative means of ribosome recruitment are not understood. Here we provide evidence that eukaryotic initiation factor (eIF) 4G controls cap-independent translation initiation at the c-myc and vascular endothelial growth factor (VEGF) 5′ UTRs in vivo. Cap-independent translation was investigated in tetracycline-inducible cell lines expressing either full-length eIF4G or a C-terminal fragment (Ct) lacking interaction with eIF4E and poly(A) binding protein. Expression of Ct, but not intact eIF4G, potently stimulated cap-independent initiation at the c-myc/VEGF 5′ UTRs. In vitro RNA-binding assays suggest that stimulation of cap-independent translation initiation by Ct is due to direct association with the c-myc/VEGF 5′ UTR, enabling 43S preinitiation complex recruitment. Our work demonstrates that variant translation initiation factors enable unconventional translation initiation at mRNA subsets with distinct structural features.     

characterization of wheat germ initiation factor eIF-2

The initiation factor eIF-2 that specifically binds Met-tRNA_f and GTP in ternary complex (eIF-2. GTP. Met-tRNA_f) has been purified to apparent homogeneity from wheat germ ribosomal salt wash. The purified factor exhibits a sedimentation coefficient of 5 · 5S and an aggregate molecular weight of 122000-daltons for the native protein.A preliminary account of this work was presented at the 66th Annual (1982) Meeting of the Federation of American Societies for Experimental Biology; Fed Proc 41, 1040.     

Eukaryotic translation initiation factor eIF5 promotes the accuracy of start codon recognition by regulating Pi release and conformational transitions of the preinitiation complex

eIF5 is the GTPase activating protein (GAP) for the eIF2·GTP·Met-tRNA_i^Met ternary complex with a critical role in initiation codon selection. Previous work suggested that the eIF5 mutation G31R/SUI5 elevates initiation at UUG codons by increasing GAP function. Subsequent work implicated eIF5 in rearrangement of the preinitiation complex (PIC) from an open, scanning conformation to a closed state at AUG codons, from which P_i is released from eIF2·GDP·P_i. To identify eIF5 functions crucial for accurate initiation, we investigated the consequences of G31R on GTP hydrolysis and P_i release, and the effects of intragenic G31R suppressors on these reactions, and on the partitioning of PICs between open and closed states. eIF5-G31R altered regulation of P_i release, accelerating it at UUG while decreasing it at AUG codons, consistent with its ability to stabilize the closed complex at UUG. Suppressor G62S mitigates both defects of G31R, accounting for its efficient suppression of UUG initiation in G31R,G62S cells; however suppressor M18V impairs GTP hydrolysis with little effect on PIC conformation. The strong defect in GTP hydrolysis conferred by M18V likely explains its broad suppression of Sui⁻ mutations in numerous factors. We conclude that both of eIF5''s functions, regulating P_i release and stabilizing the closed PIC conformation, contribute to stringent AUG selection in vivo.     

GTP interacts with the gamma-subunit of eukaryotic initiation factor eIF-2

Eukaryotic initiation factor eIF-2 is an oligomeric protein consisting of three different subunits. During initiation of protein synthesis eIF-2 interacts with GTP, Met-tRNAf and 40 S ribosomal subunit. By affinity labeling with a photo-reactive GTP analogue it was shown that in the binary complex [eIF-2 X GTP] GTP is in contact with the gamma-subunit of eIF-2.     

Formation and release of eukaryotic initiation factor 2 X GDP complex during eukaryotic ribosomal polypeptide chain initiation complex formation

The formation and release of an eukaryotic initiation factor (eIF)-2 X GDP binary complex during eIF-5-mediated assembly of an 80 S ribosomal polypeptide chain initiation complex have been studied by sucrose gradient centrifugation analysis. Isolated 40 S initiation complex reacts with eIF-5 and 60 S ribosomal subunits to form an 80 S ribosomal initiation complex with concomitant hydrolysis of an equimolar amount of bound GTP to GDP and Pi. Sucrose gradient analysis of reaction products revealed that GDP was released from ribosomes as an eIF-2 X GDP complex. Evidence is presented that eIF-5-mediated hydrolysis releases the GTP bound to the 40 S initiation complex as an intact eIF-2 X GDP complex rather than as free GDP and eIF-2 which subsequently recombine to form the binary complex. Furthermore, formation and release of eIF-2 X GDP from the ribosomal complex do not require concomitant formation of an 80 S initiation complex since both reactions occur efficiently when the 40 S initiation complex reacts with eIF-5 in the absence of 60 S ribosomal subunits. These results, along with the observation that the 40 S initiation complex formed with the nonhydrolyzable analogue of GTP, 5'-guanylylmethylene diphosphonate, can neither join a 60 S ribosomal subunit nor releases ribosome-bound eIF-2, suggest that following eIF-5-mediated hydrolysis of GTP bound to the 40 S initiation complex, both Pi and eIF-2 X GDP complex are released from ribosomes prior to the joining of 60 S ribosomal subunits to the 40 S initiation complex.     

Release and recycling of eukaryotic initiation factor 2 in the formation of an 80 S ribosomal polypeptide chain initiation complex.

The eukaryotic initiation factor (eIF)-5 mediates hydrolysis of GTP bound to the 40 S initiation complex in the absence of 60 S ribosomal subunits. The eIF-2.GDP formed under these conditions is released from the 40 S ribosomal subunit while initiator Met-tRNA(f) remains bound. The released eIF-2.GDP can participate in an eIF-2B-catalyzed GDP/GTP exchange reaction to reform the Met-tRNA(f).eIF-2.GTP ternary complex. In contrast, when 60 S ribosomal subunits were also present in an eIF-5-catalyzed reaction, the eIF-2.GDP produced remained bound to the 60 S ribosomal subunit of the 80 S initiation complex. When such an 80 S initiation complex, containing bound eIF-2.GDP, was incubated with GTP and eIF-2B, GDP was released. However, eIF-2 still remained bound to the ribosomes and was unable to form a Met-tRNA(f)l.eIF-2.GTP ternary complex. In contrast, when 60 S ribosomal subunits were preincubated with either free eIF-2 or with eIF-2.eIF-2B complex and then added to a reaction containing both the 40 S initiation complex and eIF-5, the eIF-2.GDP produced did not bind to the 60 S ribosomal subunits but was released from the ribosomes. Thus, the 80 S initiation complex formed under these conditions did not contain bound eIF-2.GDP. Under similar experimental conditions, preincubation of 60 S ribosomal subunits with purified eIF-2B (free of eIF-2) failed to cause release of eIF-2.GDP from the ribosomal initiation complex. These results suggest that 60 S ribosome-bound eIF-2.GDP does not act as a direct substrate for eIF-2B-mediated release of eIF-2 from ribosomes. Rather, the affinity of 60 S ribosomal subunits for either eIF-2, or the eIF-2 moiety of the eIF-2.eIF-2B complex, prevents association of 60 S ribosomal subunits with eIF-2.GDP formed in the initiation reaction. This ensures release of eIF-2 from ribosomes following hydrolysis of GTP bound to the 40 S initiation complex.     

A GDP/GTP exchange factor essential for eukaryotic initiation factor 2 cycling in Ehrlich ascites tumor cells and its regulation by eukaryotic initiation factor 2 phosphorylation

Formation of the ternary complex Met-tRNAi X eukaryotic initiation factor (eIF) 2 X GTP from eIF-2 X GDP requires exchange of GDP for GTP. However, at physiological Mg2+ concentrations, GDP is released from eIF-2 exceedingly slowly (Clemens, M.J., Pain, V.M., Wong, S.T., and Henshaw, E.C. (1982) Nature (Lond.) 296, 93-95). However, GDP is released rapidly from impure eIF-2 preparations, indicating the presence of a GDP/GTP exchange factor. We have now purified this factor from Ehrlich cells and refer to it as GEF. CM-Sephadex chromatography of ribosomal salt wash separated two peaks of eIF-2 activity. GEF was found in association with eIF-2 in the first peak and co-purified with eIF-2 under low salt conditions. It was separated from eIF-2 in high salt buffers and further purified on hydroxylapatite and phosphocellulose. Gel electrophoresis of our purest preparations showed major bands at 85, 67, 52, 37, 27, and 21 kDa. Purified GEF increased the rate of exchange of [32P] GDP for unlabeled GDP 25-fold but did not function with phosphorylated eIF-2 (alpha subunit). The factor also stimulated markedly the rate of ternary complex formation using eIF-2 X GDP as substrate with GTP and Met-tRNAi but not using phosphorylated eIF-2 X GDP as substrate. eIF-2 is released from the 80 S initiation complex with hydrolysis of GTP. If eIF-2 X GDP is actually the complex released, then GEF is absolutely required for eIF-2 to cycle and it is therefore a new eukaryotic initiation factor. Furthermore, the inability of GEF to utilize eIF-2 (alpha P) X GDP explains how phosphorylation of eIF-2 can inhibit polypeptide chain initiation.     

Nucleotide regulation of a eukaryotic protein synthesis initiation complex;.

Formation of a ternary initiation complex containing Met-tRNAf, GTP and eukaryotic initiation factor 2, is the first step in sequential assembly of the initiation complex. The concentration of GTP required for half maximal formation of the ternary complex is 2.5 with 10(-6) M. GDP is a potent competitive inhibitor of ternary complex formation with Ki = 3.4 with 10(-7) M. The nucleotide binding site on eukaryotic initiation factor 2 demonstrates relative specificity for GDP with KD(GDP) = 3.0 with 10(-8) M; 100-fold higher concentrations of GTP than GDP are required for displacement of either [(3)H]GDP or [(3)h]gtp from the necleotide binding site. An ATP-dependent stimulation of ternary complex formation observed in partially purified initiation factor preparations is due to nucleoside diphosphate kinase (EC 2.7.4.6) which serves to remove inhibitory levels of GDP by phosphorylation with ATP. Since GTP is hydrolyzed to GDP during protein synthesis, this provides a mechanism by which the ATP:ADP ratio may regulate the rate of initiation of protein synthesis.     

The archaeal eIF2 homologue: functional properties of an ancient translation initiation factor

The eukaryotic translation initiation factor 2 (eIF2) is pivotal for delivery of the initiator tRNA (tRNAi) to the ribosome. Here, we report the functional characterization of the archaeal homologue, a/eIF2. We have cloned the genes encoding the three subunits of a/eIF2 from the thermophilic archaeon Sulfolobus solfataricus, and have assayed the activities of the purified recombinant proteins in vitro. We demonstrate that the trimeric factor reconstituted from the recombinant polypeptides has properties similar to those of its eukaryal homologue: it interacts with GTP and Met-tRNAi, and stimulates binding of the latter to the small ribosomal subunit. However, the archaeal protein differs in some functional aspects from its eukaryal counterpart. In contrast to eIF2, a/eIF2 has similar affinities for GDP and GTP, and the β-subunit does not contribute to tRNAi binding. The detailed analysis of the complete trimer and of its isolated subunits is discussed in light of the evolutionary history of the eIF2-like proteins.     

Identification of ribosome-bound eukaryotic initiation factor 2.GDP binary complex as an intermediate in polypeptide chain initiation reaction

Studies on the formation and release of the eukaryotic initiation factor (eIF)-2.GDP binary complex formed during eIF-5-mediated assembly of an 80 S initiation complex have been carried out. Incubation of a 40 S initiation complex with eIF-5, in the presence or absence of 60 S ribosomal subunits at 25 degrees C, causes rapid and quantitative hydrolysis of ribosome-bound GTP to form an eIF-2.GDP binary complex and Pi. Analysis of both reaction products by Sephadex G-200 gel filtration reveals that while Pi is released from ribosomes, the eIF-2.GDP complex remains bound to the ribosomal initiation complex. The eIF-2.GDP binary complex can however be released from ribosome by subjecting the eIF-5-catalyzed reaction products to either longer periods of incubation at 37 degrees C or sucrose gradient centrifugation. Furthermore, addition of a high molar excess of isolated eIF-2.GDP binary complex to a 40 S initiation reaction mixture does not cause exchange of ribosome-bound eIF-2.GDP complex formed by eIF-5-catalyzed hydrolysis of GTP. These results indicate that eIF-2.GDP complex is directly formed on the surface of ribosomes following hydrolysis of GTP bound to a 40 S initiation complex, and that ribosome-bound eIF-2 X GDP complex is an intermediate in polypeptide chain initiation reaction.     

The ribosome‐bound initiation factor 2 recruits initiator tRNA to the 30S initiation complex

Bacterial translation initiation factor 2 (IF2) is a GTPase that promotes the binding of the initiator fMet‐tRNA^fMet to the 30S ribosomal subunit. It is often assumed that IF2 delivers fMet‐tRNA^fMet to the ribosome in a ternary complex, IF2·GTP·fMet‐tRNA^fMet. By using rapid kinetic techniques, we show here that binding of IF2·GTP to the 30S ribosomal subunit precedes and is independent of fMet‐tRNA^fMet binding. The ternary complex formed in solution by IF2·GTP and fMet‐tRNA is unstable and dissociates before IF2·GTP and, subsequently, fMet‐tRNA^fMet bind to the 30S subunit. Ribosome‐bound IF2 might accelerate the recruitment of fMet‐tRNA^fMet to the 30S initiation complex by providing anchoring interactions or inducing a favourable ribosome conformation. The mechanism of action of IF2 seems to be different from that of tRNA carriers such as EF‐Tu, SelB and eukaryotic initiation factor 2 (eIF2), instead resembling that of eIF5B, the eukaryotic subunit association factor.     

Structural dynamics of bacterial translation initiation factor IF2

Bacterial translation initiation factor IF2 promotes ribosomal subunit association, recruitment, and binding of fMet-tRNA to the ribosomal P-site and initiation dipeptide formation. Here, we present the solution structures of GDP-bound and apo-IF2-G2 of Bacillus stearothermophilus and provide evidence that this isolated domain binds the 50 S ribosomal subunit and hydrolyzes GTP. Differences between the free and GDP-bound structures of IF2-G2 suggest that domain reorganization within the G2-G3-C1 regions underlies the different structural requirements of IF2 during the initiation process. However, these structural signals are unlikely forwarded from IF2-G2 to the C-terminal fMet-tRNA binding domain (IF2-C2) because the connected IF2-C1 and IF2-C2 modules show completely independent mobility, indicating that the bacterial interdomain connector lacks the rigidity that was found in the archaeal IF2 homolog aIF5B.