Evidence that the 59-kDa protein synthesis initiation factor from wheat germ is functionally similar to the 80-kDa initiation factor 4B from mammalian cells

The results of this investigation show that the 59-kDa protein synthesis initiation factor from wheat germ, designated eukaryotic initiation factor (eIF)-4G by Browning et al. (Browning, K.S., Maia, D.M., Lax, S.R., and Ravel, J.M. (1987) J. Biol. Chem. 262, 539-541), cross-links to the 5'-terminal cap of oxidized mRNA in the presence of eIF-4A, eIF-4F, and ATP, stimulates the RNA-dependent ATPase activities of eIF-4A and a mixture of eIF-4A and eIF-4F, and stimulates the unwinding activities of eIF-4A, eIF-4F, and a mixture of eIF-4A and eIF-4F. These findings strongly suggest that the 59-kDa factor from wheat germ is the functional equivalent of the 80-kDa protein synthesis initiation factor, eIF-4B, from mammalian cells. Recent reports indicate that the wheat germ initiation factor which contains two subunits of 80 and 28 kDa and which was given the designation "eIF-4B" by Lax et al. (Lax, S.R., Lauer, S.J., Browning, K. S., and Ravel, J.M. (1986) Methods Enzymol. 118, 109-128) is an isozyme form of eIF-4F and not the functional equivalent of mammalian eIF-4B. On the basis of functional characteristics we propose that the designation for the wheat germ factor containing the 80- and 28-kDa polypeptides be changed from eIF-4B to eIF-(iso)4F and the designation for the 59-kDa factor be changed from eIF-4G to eIF-4B.     

Phosphorylation of wheat germ initiation factors and ribosomal proteins

The ability of the wheat germ initiation factors and ribosomes to serve as substrates for a wheat germ protein kinase (Yan and Tao 1982 J Biol Chem 257: 7037-7043) has been investigated. The wheat germ kinase catalyzes the phosphorylation of the 42,000 dalton subunit of eukaryotic initiation factor (eIF)-2 and the 107,000 dalton subunit of eIF-3. Other initiation factors, eIF-4B and eIF-4A, and elongation factors, EF-1 and EF-2, are not phosphorylated by the kinase. Quantitative analysis indicates that the kinase catalyzes the incorporation of about 0.5 to 0.6 mole of phosphate per mole of the 42,000 dalton subunit of eIF-2 and about 6 moles of phosphate per mole of the 107,000 dalton subunit of eIF-3. Three proteins (M_r = 38,000, 14,800, and 12,600) of the 60S ribosomal subunit are phosphorylated by the kinase, but none of the 40S ribosomal proteins are substrates of the kinase. No effects of phosphorylation on the activities of eIF-2, eIF-3, or 60S ribosomal subunits could be demonstrated in vitro.     

Identification of an isozyme form of protein synthesis initiation factor 4F in plants.

We showed previously that wheat germ extracts contain two forms of protein synthesis initiation factor 4F that have very similar functional properties (Browning, K. S., Lax, S. R., and Ravel, J. M. (1987) J. Biol. Chem. 262, 11228-11232). One form, designated eIF-4F, is a complex containing two subunits, p220 and p26. The other form, designated eIF-(iso)4F, is a complex containing two subunits, p82 and p28, which are antigenically distinct from the subunits of eIF-4F. Both the p26 subunit of eIF-4F and the p28 subunit of eIF-(iso)4F are m7G cap-binding proteins. In this investigation, affinity-purified antibodies to the p220 and p26 subunits of wheat germ eIF-4F and to the p82 and p28 subunits of wheat germ eIF-(iso)4F were used to determine if isozyme forms of eIF-4F are present in maize and cauliflower. Extracts from wheat germ, maize root tips, and cauliflower inflorescences were partially purified by adsorption on m7GTP-Sepharose and elution with m7GTP (MGS eluate). Analysis by sodium dodecyl sulfate gel electrophoresis and immunoblotting with antibodies to the subunits of the wheat germ factors showed that the MGS eluate from maize contains polypeptides that react with antibodies to the p82 and p28 subunits of wheat eIF-(iso)4F, as well as polypeptides that react with antibodies to the p220 and p26 subunits of wheat eIF-4F. The MGS eluate from cauliflower also contains polypeptides that reacted with antibodies to the subunits of wheat eIF-(iso)4F. These results indicate that both maize and cauliflower contain the isozyme form of eIF-4F. In addition, it was found that the factors in the MGS eluate from maize support polypeptide synthesis in a system from wheat deficient in eIF-4F and eIF-(iso)4F, whereas the factors in the MGS eluate from cauliflower support polypeptide synthesis only to a small extent.     

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.     

Comparative analysis of phosphorylation of translational initiation and elongation factors by seven protein kinases

Four initiation factors (eIF-2, -3, -4B, and -4F), previously shown to be phosphorylated in vivo, are each phosphorylated to a significant extent in vitro (greater than 0.3 mol of phosphate/mol of factor) by at least three different protein kinases. An S6 kinase from liver, an active form of protease-activated kinase II which modifies the same sites on S6 as those phosphorylated in vivo in response to mitogens, phosphorylates the beta subunit of eIF-2, eIF-3 (p120-p130), eIF-4B, and eIF-4F (p220). The Ca2+, phospholipid-dependent protein kinase phosphorylates eIF-2 beta, eIF-3 (p170, p120-p130), eIF-4B, and eIF-4F (p220, p25). The cAMP-dependent protein kinase significantly modifies eIF-4B and, to a lesser extent, eIF-3 (p130). Casein kinase I incorporates phosphate only into eIF-4B, but to a limited extent. Casein kinase II phosphorylates eIF-2 beta, eIF-3 (p170, p120), and eIF-4B, while protease-activated kinase I modifies eIF-3 (p170, p120-p130), eIF-4B, and eIF-4F (p220). The mitogen-stimulated S6 kinase from 3T3-L1 cells, activated in response to insulin, does not phosphorylate any of the initiation factors. There is no significant incorporation of phosphate into eIF-2 alpha or -gamma, eIF-4A, eIF-4C, eIF-4D, EF-1, or EF-2 by any of the protein kinases examined. Phosphopeptide mapping of tryptic digests of the phosphorylated subunits shows that the individual protein kinases modify different sites. The sites phosphorylated in vitro reflect those modified in vivo as shown with eIF-4F in concomitant studies with reticulocytes treated with tumor-promoting phorbol ester (Morley, S.J., and Traugh, J. A. J. Biol. Chem., in press). Thus, we have identified multipotential protein kinases which modify four initiation factors phosphorylated in vivo and have shown that phosphorylation of these translational components can be coordinately regulated.     

Evidence that the primary effect of phosphorylation of eukaryotic initiation factor 2(alpha) in rabbit reticulocyte lysate is inhibition of the release of eukaryotic initiation factor-2.GDP from 60 S ribosomal subunits

The phosphorylation of eukaryotic initiation factor (eIF) 2 alpha that occurs when rabbit reticulocyte lysate is incubated in the absence of hemin or with poly(I.C) causes inhibition of polypeptide chain initiation by preventing a separate factor (termed RF) from promoting the exchange of GTP for GDP on eIF-2. When lysate was incubated in the presence of hemin and [14C] eIF-2 or [alpha-32P]GTP, we observed binding of eIF-2 and GDP or GTP to 60 S ribosomal subunits that was slightly greater than that bound to 40 S subunits and little binding to 80 S ribosomes. When incubation was in the absence of hemin or in the presence of hemin plus 0.1 microgram/ml poly(I.C), eIF-2 and GDP binding to 60 S subunits was increased 1.5- to 2-fold, that bound to 80 S ribosomes was almost as great as that bound to 60 S subunits, and that bound to 40 S subunits was unchanged. Our data indicate that about 40% of the eIF-2 that becomes bound to 60 S subunits and 80 S ribosomes in the absence of hemin or with poly(I.C) is eIF-2(alpha-P) and suggest that the eIF-2 and GDP bound is probably in the form of a binary complex. The accumulation of eIF-2.GDP on 60 S subunits occurs before binding of Met-tRNAf to 40 S subunits becomes reduced and before protein synthesis becomes inhibited. The rate of turnover of GDP (presumably eIF-2.GDP) on 60 S subunits and 80 S ribosomes in the absence of hemin is reduced to less than 10% the control rate, because the dissociation of eIF-2.GDP is inhibited. Additional RF increases the turnover of eIF-2.GDP on 60 S subunits and 80 S ribosomes to near the control rate by promoting dissociation of eIF-2.GDP but not eIF-2(alpha-P).GDP. Our findings suggest that eIF-2.GTP binding to and eIF-2.GDP release from 60 S subunits may normally occur and serve to promote subunit joining. The phosphorylation of eIF-2 alpha inhibits polypeptide chain initiation by preventing dissociation of eIF-2.GDP from either free 60 S subunits (thus inhibiting subunit joining directly) or the 60 S subunit component of an 80 S initiation complex (thereby blocking elongation and resulting in the dissociation of the 80 S complex).     

Activity of Euglena gracilis chloroplast ribosomes with procaryotic and eucaryotic initiation factors

A method that permits the preparation of Euglena gracilis chloroplast 30 S ribosomal subunits that are largely free of endogenous initiation factors and that are active in the binding of fMet-tRNA in response to poly(A, U, G), has been developed. These 30 S subunits have been tested for activity in initiation complex formation with initiation factors from both procaryotes and eucaryotes. We have observed that Escherichia coli IF-2 binds fMet-tRNA nearly as well to Euglena chloroplast ribosomal subunits as it does to its homologous subunits. Neither wheat germ eIF-2 nor Euglena eIF-2A can bind fMet-tRNA efficiently to Euglena chloroplast or E. coli 30 S subunits although both are active with wheat germ 40 S ribosomal subunits. Euglena chloroplast 68 S ribosomes will also bind the initiator tRNA. Both E. coli IF-2 and E. coli IF-3 stimulate this reaction on chloroplast ribosomes with approximately the same efficiency as they do on their homologous ribosomes. E. coli IF-1 enhances the binding of fMet-tRNA to the chloroplast 68 S ribosomes when either IF-2 or IF-3 is limiting. The chloroplast ribosomes unlike E. coli ribosomes show considerable activity over a broad range of Mg2+ ion concentrations.     

Immunofluorescent localization of protein synthesis components in mouse embryo fibroblasts

The distribution of initiation factor 2(eIF-2) and elongation factor 2(EF-2) in cultured mouse embryo fibroblasts was studied and compared with the distribution of ribosomes. We used immunofluorescence microscopy with monospecific antibodies to eIF-2, EF-2, and proteins S3a and S7 of the small ribosomal subunit. Ribosomes and factors eIF-2 and EF-2 were found mainly in the vicinity of the cell nucleus. This perinuclear zone coincides with the endoplasm - the central part of the cell containing numerous membraneous organelles and inclusions. Besides the perinuclear zone, small stained regions could be seen at the periphery of some cells. After treatment of the cells with Triton X-100 in a buffer conditions, that stabilizes the major cytoskeletal structures, some of the ribosomes, eIF-2, and EF-2 remained bound to the insoluble material. These components were found near the nucleus and some were located along the microfilament bundles.     

Evidence that the requirements for ATP and wheat germ initiation factors 4A and 4F are affected by a region of satellite tobacco necrosis virus RNA that is 3' to the ribosomal binding site

A cDNA containing the complete genome of satellite tobacco necrosis virus (STNV) RNA was constructed and cloned into a plasmid vector containing the T7 polymerase promotor. A second clone containing the first 54 nucleotides from the 5' end, which includes the ribosome binding site, was also constructed. RNAs were transcribed from these plasmids (pSTNV1239 and pSTNV54) and tested for their ability to bind to wheat germ 40 S ribosomal subunits in the presence of wheat germ initiation factors eIF-4A, eIF-4F, eIF-4G, eIF-3, eIF-2, Met-tRNA, ATP, and guanosine 5'-(beta, gamma-imino)triphosphate (GMP-PNP). Maximal binding of the STNV RNA transcribed from pSTNV1239 is obtained only in the presence of all the initiation factors and ATP. In contrast, close to maximal binding of STNV RNA transcribed from pSTNV54 is obtained in the absence of eIF-4A, eIF-4F, eIF-4G, and ATP. A series of deletion clones from the 3' end of the STNV cDNA was prepared, and the requirements for binding to 40 S ribosomal subunits were determined. STNV RNAs containing more than 134 nucleotides from the 5' end require eIF-4A, eIF-4F, eIF-4G, and ATP for maximal binding to 40 S ribosomal subunits, whereas STNV RNAs containing 86 nucleotides or less no longer require ATP and these factors. These findings indicate that a region 3' to the initiation codon affects the requirements for eIF-4A, eIF-4F, eIF-4G, and ATP.     

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.     

Evidence that the 5'-untranslated leader of mRNA affects the requirement for wheat germ initiation factors 4A, 4F, and 4G

The efficiency of translation of alfalfa mosaic virus (AMV) RNA 4, barley alpha-amylase (B alpha A) mRNA, and two chimeric mRNAs, AMV 4-B alpha A and B alpha A-AMV 4 (in which the 5' leader sequences of the two mRNAs were interchanged), was measured in an S30 extract from wheat germ and a fractionated system from wheat germ in which translation could be made dependent upon initiation factor (eIF) 3, 4A, 4F, or 4G. In the S30 system, AMV RNA 4 and the chimeric mRNA AMV 4-B alpha A are translated much more efficiently than B alpha A mRNA and the chimeric mRNA B alpha A-AMV 4. When the S30 system was supplemented with high amounts of purified eIF-3, eIF-4A, eIF-4F, and eIF-4G, B alpha A and B alpha A-AMV 4 mRNAs were translated as efficiently as AMV RNA 4 and AMV 4-B alpha A mRNA. These findings indicated that the mRNAs containing the B alpha A leader sequence required higher amounts of one or more of the initiation factors (eIF-3, eIF-4A, eIF-4F, and eIF-4G) for efficient translation. Determination of the amounts of the initiation factors required for translation in the fractionated system showed that AMV RNA 4 required 2-4-fold lower amounts of eIF-3, eIF-4A, eIF-4F, and eIF-4G than did B alpha A mRNA. Replacement of the B alpha A leader sequence with that of AMV RNA 4 decreased the amounts of eIF-4A, eIF-4G, and eIF-3 required, but did not affect the amount of eIF-4F required. Replacement of the AMV RNA 4 leader sequence with that of B alpha A mRNA increased the amounts of eIF-4F, eIF-4G, and eIF-3 required, but did not affect the amount of eIF-4A required. These data strongly suggest that the amounts of the factors required are affected not only by the 5' leader itself but also by interactions between the 5' leader and a region(s) of the mRNA 3' to the initiation codon.     

Function of eukaryotic initiation factor 5 in the formation of an 80 S ribosomal polypeptide chain initiation complex.

Eukaryotic initiation factor 5 (eIF-5), isolated from rabbit reticulocyte lysates, is a monomeric protein of 58-62 kDa. The function of eIF-5 in the formation of an 80 S polypeptide chain initiation complex from a 40 S initiation complex has been investigated. Incubation of the isolated 40 S initiation complex (40 S.AUG.Met.tRNAf.eIF-2 GTP) with eIF-5 resulted in the rapid and quantitative hydrolysis of GTP bound to the 40 S initiation complex. The rate of this reaction was unaffected by the presence of 60 S ribosomal subunits. Analysis of eIF-5-catalyzed reaction products by gel filtration indicated that both eIF-2.GDP binary complex and Pi formed were released from the ribosomal complex whereas Met-tRNAf remained bound to 40 S ribosomes as a Met-tRNAf.40 S.AUG complex. Reactions carried out with biologically active 32P-labeled eIF-5 indicated that this protein was not associated with the 40 S.AUG.Met-tRNAf complex; similar results were obtained by immunological methods using monospecific anti-eIF-5 antibodies. The isolated 40 S.AUG.Met-RNAf complex, free of eIF-2.GDP binary complex and eIF-5, readily interacted with 60 S ribosomal subunits in the absence of exogenously added eIF-5 to form the 80 S initiation complex capable of transferring Met-tRNAf into peptide linkages. These results indicate that the sole function of eIF-5 in the initiation of protein synthesis is to mediate hydrolysis of GTP bound to the 40 S initiation complex in the absence of 60 S ribosomal subunits. This leads to formation of the intermediate 40 S.AUG.Met-tRNAf and dissociation of the eIF-2.GDP binary complex. Subsequent joining of 60 S ribosomal subunits to the intermediate 40 S.AUG.Met-tRNAf complex does not require participation of eIF-5. Thus, the formation of an 80 S ribosomal polypeptide chain initiation complex from a 40 S ribosomal initiation complex can be summarized by the following sequence of partial reactions. (40 S.AUG.Met-tRNAf.eIF-2.GTP) eIF-5----(40 S.AUG.Met-tRNAf) + (eIF-2.GDP) + Pi (1) (40 S.AUG.Met-tRNAf) + 60 S----(80 S.AUG.Met-tRNAf) (2) 80 S initiation complex.     

Initiation of mammalian protein synthesis. I. Purification and characterization of seven initiation factors

We have purified seven protein factors from rabbit reticulocytes, all of which are presumed to be involved in the initiation of mammalian protein synthesis. They are termed eIF-1, eIF-2, eIF-3, eIF4A, eIF-4B, eIF-4C and e-IF-5. The purification from the KCl wash of crude ribosomes involves fractionation with ammonium sulphate, ion-exchange chromatography and separation by size. The operational definition of an initiation factor was its requirement for translation of natural messenger RNA (globin mRNA) in a highly purified and fractionated system using completely defined elongation components, i.e. aminoacyl-tRNA, the two elongation factors EF-1 and EF-2, and GTP. By the same criterion ATP was also shown to be required for initiation. The initiation factors were purified to homogeneity with the exception of eIF-4B, which was 60% to 70% pure. They were characterized physically by sucrose gradient centrifugation and by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. With the exception of eIF-2 and eIF-3, they consist of single polypeptide chains ranging in molecular weight from 15,000 (eIF-1) to about 160,000 (eIF-5). The factor eIF-2 has three subunits of about 35,000, 50,000 and 55,000 molecular weight. The factor eIF-3 appears to be homogeneous as judged by gel electrophoresis in non-dissociating conditions and sedimentation analysis. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate, however, reveals at least nine subunits ranging in molecular weight from about 35,000 to 160,000. Initiation complexes (mRNA · Met-tRNA_f · 80 S ribosome), made in the presence of the seven initiation factors, ATP and GTP were isolated on a sucrose gradient and shown to be fully active in polypeptide chain elongation when supplied with aminoacyl-tRNA, the two elongation factors and GTP.     

Binding and release of radiolabeled eukaryotic initiation factors 2 and 3 during 80 S initiation complex formation.

The AUG-dependent formation of an 80 S ribosomal initiation complex was studied using purified rabbit reticulocyte initiation factors radiolabeled by reductive methylation. The radiolabeled initiation factors were as biologically active as untreated factors. Reaction mixtures containing a variety of components (AUG, GTP, Met-tRNAf, initiation factors, and 40 S and 60 S ribosomal subunits) were incubated at 30 degrees C and then analyzed on linear sucrose gradients for the formation of ribosomal complexes. The results show that both eukaryotic initiation factor (eIF)-3 and the ternary complex (eIF-2.GTP.Met-tRNAf) bind independently to the 40 S subunit and each of these components enhances the binding of the other. All of the polypeptides of eIF-2 and eIF-3 participate in this binding. Formation of an 80 S ribosomal complex requires eIF-5 and 60 S subunits in a reaction that is stimulated by eIF-4C. Both eIF-2 and eIF-3 are released from the 40 S preinitiation complex during formation of the 80 S initiation complex. Release of eIF-2 and eIF-3 does not occur and 80 S ribosomal complexes are not formed if GTP is replaced by a nonhydrolyzable analog such as guanosine 5'-O3-(1,2-mu-imido)triphosphate. Despite a variety of attempts, it has not yet been possible to demonstrate binding of eIF-4C, eIF-4D, or eIF-5 to either 40 S or 80 S ribosomal complexes.     

The effect of elevated temperature on protein synthesis in cell-free extracts of cultured Chinese hamster ovary cells

The effect of elevated temperature on the activity of various components involved in protein synthesis was investigated in extracts from cultured Chinese hamster ovary cells. The translation of exogenous mRNA was markedly inhibited by preincubation of the extract for 15 to 20 minutes at 42°C. However, the following intermediary reactions were not affected, or only slightly inhibited, at 42°C: 1) the incorporation of Met-tRNA_f into eIF-2·Met-tRNA_f·GTP ternary complex; 2) the interaction of the ternary complex with 40S ribosomal subunits to form the 40S preinitiation intermediate; 3) the binding of mRNA and 60S subunits to form the 80S initiation complex; and 4) the reactions catalyzed by elongation factors EF-1 and EF-2. The activity of Met-tRNA synthetase was markedly inhibited, affecting the formation of initiator Met-tRNA_f required for the initiation of protein synthesis and the translation of natural mRNA. Other aminoacyl-tRNA synthetases were not significantly affected by the elevated temperature.     

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.     

Identification of two messenger RNA cap binding proteins in wheat germ. Evidence that the 28-kDa subunit of eIF-4B and the 26-kDa subunit of eIF-4F are antigenically distinct polypeptides

Previous work has shown that eukaryotic initiation factor (eIF)-4B from wheat germ is a complex containing two subunits, 80 and 28 kDa, and eIF-4F from wheat germ is a complex containing two subunits, 220 and 26 kDa (Lax, S., Fritz, W., Browning, K., and Ravel, J. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 330-333). Here we show that both the 28-kDa subunit of eIF-4B and the 26-kDa subunit of eIF-4F cross-link to the 5' terminus of capped and oxidized satellite tobacco necrosis virus RNA in the absence of ATP and that the cross-linking of both polypeptides is inhibited by m7GDP. Several lines of evidence indicate that the 28-kDa and the 26-kDa cap binding proteins of eIF-4B and eIF-4F are antigenically distinct polypeptides. Rabbit polyclonal antibodies raised to intact eIF-4B or to the isolated 28-kDa subunit of eIF-4B react strongly with the 28-kDa subunit of eIF-4B on immunoblots, but show only a very weak reaction with the 26-kDa subunit of eIF-4F under the same conditions. In addition, a mouse monoclonal antibody was obtained that reacts strongly with the 26-kDa subunit of eIF-4F but does not react with the 28-kDa subunit of eIF-4B. Evidence is presented also which indicates that the higher molecular weight subunits of eIF-4B and eIF-4F are antigenically distinct. Rabbit polyclonal antibodies raised to intact eIF-4B or the isolated 80-kDa subunit inhibit eIF-4B-dependent polypeptide synthesis but do not inhibit eIF-4F-dependent polypeptide synthesis. Rabbit polyclonal antibodies raised to eIF-4F inhibit eIF-4F-dependent polypeptide synthesis but do not inhibit eIF-4B-dependent polypeptide synthesis.     

Evidence that phosphorylation of eIF-2(alpha) prevents the eIF-2B-mediated dissociation of eIF-2 X GDP from the 60 S subunit of complete initiation complexes

Recent observations have indicated that eukaryotic initiation factor (eIF)-2 and GTP or GDP normally bind to 60 S ribosomal subunits in rabbit reticulocyte lysate and that when eIF-2 alpha is phosphorylated and polypeptide chain initiation is inhibited, eIF-2 X GDP accumulates on 60 S subunits due to impaired dissociation that is normally mediated by the reversing factor (eIF-2B). Current findings now indicate that inhibition due to phosphorylation of eIF-2 alpha is mediated, at least in part, by the inability to dissociate eIF-2 X GDP from the 60 S subunit of complete initiation complexes. At the onset of inhibition, there is an accumulation of Met-tRNA(f) and eIF-2 on the polysomes, despite a marked reduction in Met-tRNA(f) bound to 40 S subunits and Met-peptidyl-tRNA bound to the polysomes. This initial effect is not associated with the formation of "half-mers" (polysomes containing an extra unpaired 40 S subunit), and the 40 S X Met-tRNA(f) complexes, though reduced, still sediment at 43 S. When inhibition is maximal and the polysomes are largely disaggregated, there is an accumulation of 48 S complexes consisting of a 40 S subunit and Met-tRNA(f) bound to globin mRNA as well as small polysomal half-mers, such that residual protein synthesis occurs to about the same degree on "1 1/2"s and "2 1/2"s as on mono-, di-, and triribosomes. Exogenous eIF-2B increases protein synthesis on mono-, di-, and triribosomes and decreases that on half-mers. This is associated with reduced binding of Met-tRNA(f) and eIF-2 to ribosomal particles sedimenting at 80 S and greater and a shift from 48 S to 43 S complexes. These results suggest that eIF-2B must normally promote dissociation of eIF-2 X GDP from the 60 S subunit of complete initiation complexes before they can elongate but cannot when eIF-2 alpha is phosphorylated, resulting in the accumulation of these complexes, some of which dissociate into Met-tRNA(f) X 40 S X mRNA and 60 S X eIF-2 X GDP.     

Identification of proteins of the 40 S ribosomal subunit involved in interaction with initiation factor eIF-2 in the quaternary initiation complex by means of monospecific antibodies

Monospecific polyclonal antibodies against seven proteins of the 40 S subunit of rat liver ribosomes were used to identify ribosomal proteins involved in interaction with initiation factor eIF-2 in the quaternary initiation complex [eIF-2 X GMPPCP X [3H]Met-tRNAf X 40 S ribosomal subunit]. Dimeric immune complexes of 40 S subunits mediated by antibodies against ribosomal proteins S3a, S13/16, S19 and S24 were found to be unable to bind the ternary initiation complex [eIF-2 X GMPPCP X [3H]Met-tRNAf]. In contrast, 40 S dimers mediated by antibodies against proteins S2, S3 and S17 were found to bind the ternary complex. Therefore, from the ribosomal proteins tested, only proteins S3a, S13/16, S19 and S24 are concluded to be involved in eIF-2 binding to the 40 S subunit.     

Phosphorylation of eIF-4F by protein kinase C or multipotential S6 kinase stimulates protein synthesis at initiation

Eukaryotic initiation factor (eIF) 4F, a multiprotein cap binding complex, has been shown to be phosphorylated in vivo in response to phorbol 12-myristate 13-acetate and insulin (Morley, S.J., and Traugh, J.A. (1990) J. Biol. Chem. 264, 2401-2404; Morley, S.J., and Traugh, J.A. (1990) J. Biol. Chem. 265, 10611-10616). The effect of phosphorylation on the activity of purified eIF-4F, utilizing both protein kinase C and a multifunctional S6 kinase, previously identified as protease activated kinase II, has been examined; these protein kinases modify eIF-4F p25 and p220 and eIF-4F p220, respectively. Studies with an eIF-4F-dependent protein synthesis system showed that phosphorylation of eIF-4F with either protein kinase resulted in a 3-5-fold stimulation of translation relative to the nonphosphorylated control. Chemical cross-linking of eIF-4F to cap-labeled mRNA, showed that phosphorylation increased the interaction of both the p25 and p220 subunits of eIF-4F with the 5' end of mRNA. This effect was manifested by a stimulation of initiation complex formation as measured by an increase in the association of labeled mRNA with 40 S ribosomal subunits in the translation system. Thus, phosphorylation of eIF-4F enhances binding to mRNA, resulting in a stimulation of protein synthesis at initiation.