Characterization of eukaryotic initiation factor 5 from rabbit reticulocytes. Evidence that the initiation factor is a monomeric protein of Mr of about 58,000-62,000

Eukaryotic initiation factor 5 (eIF-5) has been purified from the ribosomal salt-wash proteins of rabbit reticulocyte lysates. The purified factor migrates as a single polypeptide upon sodium dodecyl sulfate-gel electrophoresis with an apparent Mr of about 58,000-62,000. In contrast, less pure preparations of reticulocyte eIF-5 behave in gel filtration columns and in glycerol gradient centrifugation in buffers containing 75-100 mM KCl as a protein of apparent Mr = 140,000-160,000. Presumably, this is due to association of the factor with other proteins, since eIF-5 activity present in such preparations can also be shown by (a) glycerol gradient centrifugation in buffers containing 500 mM KCl or (b) gel electrophoresis under denaturing conditions, to be associated with a 58,000-62,000-dalton protein. Furthermore, eIF-5 purified from rabbit reticulocyte lysates in the absence or presence of protease inhibitors is indistinguishable with regard to molecular weight and final specific activity. It can be calculated that 1 pmol of the purified eIF-5 catalyzes the formation of nearly 50 pmol of 80 S initiation complex under in vitro initiation reaction conditions. Because of the highly catalytic activity of eIF-5 in initiation reactions, the presence of even low levels of eIF-5 in eIF-2 preparations causes hydrolysis of GTP bound to the 40 S initiation complex. This results in destabilization of Met-tRNA(f) bound to the 40 S complex in sucrose gradient centrifugation.     

Eukaryotic initiation factor 5 from calf liver is a single polypeptide chain protein of Mr = 62,000

Eukaryotic initiation factor 5 (eIF-5), which specifically catalyzes the joining of a 60 S ribosomal subunit to a 40 S initiation complex to form a functional 80 S initiation complex, has been purified from ribosomal salt wash proteins of calf liver. The purified factor exhibits only one polypeptide band of Mr = 62,000 following electrophoresis in 10% polyacrylamide gels in the presence of sodium dodecyl sulfate. The native protein has a sedimentation coefficient of 4.2 S and a Stokes radius of 33 A which is consistent with eIF-5 being a monomeric protein of Mr = 58,000-62,000. Less pure preparations of eIF-5 elute in gel filtration columns with an apparent Mr of 160,000-180,000 presumably due to association of eIF-5 with other high molecular weight proteins since eIF-5 activity present in such preparations can also be shown by gel electrophoretic separation under denaturing conditions to be associated with a 62,000-dalton protein. Furthermore, eIF-5 purified from calf liver extracts with or without a number of protease inhibitors is indistinguishable with regard to molecular weight and final specific activity of purified preparations. The purified factor catalyzes the hydrolysis of GTP present in 40 S initiation complexes in the absence of 60 S ribosomal subunits. The presence of 60 S ribosomal subunits neither stimulates nor inhibits the hydrolysis of GTP. However, the factor cannot mediate 40 S or 40 + 60 S ribosome-dependent hydrolysis of GTP in the absence of Met-tRNAf or other components required for 40 S initiation complex formation. It can be calculated that 1 pmol of eIF-5 protein can catalyze the formation of at least 10 pmol of 80 S initiation complex under the conditions of in vitro initiation reactions.     

A new purification scheme for elongation factor 1 from rabbit reticulocytes and investigation of the homology of the subunits with those of initiation factor 2.

The aim of this work was to compare the subunits of the elongation factor EF-1 and the initiation factor eIF-2 from rabbit reticulocytes. We devised a simple procedure for the purification of EF-1: stepwise chromatography on heparin-Sepharose, separation of the heavy form by sucrose gradient centrifugation, and a final step of stepwise chromatography on RNA-Sepharose. The heparin-Sepharose column also clearly separated EF-1 and EF-2 within one chromatographic step. The EF-1 was 350-fold puried and the yield was 10%. This preparation showed after electrophoresis on polyacylamide gels in the presence of sodium dodecyl sulfate three bands corresponding to those described by others as the subunits, with Mr of 54000, 49000 and 29200. An additional band of Mr 34000 was present but no others. The 49000-Mr and 34000-Mr bands corresponded exactly in molecular weight to two of three subunits of eIF-2. A more detailed comparison was therefore made of all subunits of EF-1 and eIF-2. This was done by examination of chymotryptic fingerprints on polyacrylamide gel electrophoresis. No evidence for homology between EF-1 and eIF-2 was found. However, the two larger subunits of eIF-2 had a majority of chymotryptic fragments in common, thus indicating some homology between these polypeptides.     

Purified eukaryotic initiation factor 2 from calf liver consists of two polypeptide chains of 48,000 and 38,000 daltons.

Eukaryotic initiation factor 2 (eIF-2), which specifically binds Met-tRNAMetf and forms stable ternary complexes with GTP, has been purified from ribosomal salt wash proteins from calf liver. The purified factor exhibits only two polypeptide bands of Mr = 48,000 and 38,000 following electrophoresis in 15% polyacrylamide gels in the presence of sodium dodecyl sulfate. Densitometric tracings show the two polypeptides are present in a molar ratio of 1:1. This suggests a Mr = 86,000 for the native enzyme, a value which agrees with the apparent molecular weight determined by physical methods. Less pure preparations of eIF-2 show additional polypeptide bands, including 50,000- and 46,000-dalton bands, all of which can be removed by further purification without affecting the activity of eIF-2.     

Protein synthesis initiation factor 2 from chicken reticulocytes: purification by affinity chromatography and preliminary characterization

Affinity chromatography on beta,gamma-methylene guanosine 5'-triphosphate-Sepharose was used to purify protein synthesis initiation factor eIF-2 from chicken reticulocytes. Gel filtration of the purified factor gave a molecular weight of 150,000, whereas electrophoresis of the purified factor on polyacrylamide gel containing sodium dodecyl sulfate revealed three non-identical subunits with apparent molecular weight of 57,000, 41,000 and 33,000. With Met-tRNA and GTP, the factor formed a ternary complex which would bind the 40S ribosomal subunits. Treatment of the factor with N-ethylmaleimide resulted in a loss of activity. Two sulfhydryl groups per eIF-2 molecule were essential for activity.     

Studies of a factor from dystrophic mouse muscle inhibitory towards protein synthesis.

A substance inhibitory to protein synthesis was purified from mouse skeletal muscle by gel filtration and ion-exchange chromatography, as well as by centrifugation on sucrose gradients. The molecular weight of the inhibitor, determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, was 71000. The inhibitory activity was insensitive to ribonuclease A, deoxyribonuclease I and phospholipase C. It was sensitive to Pronase treatment but insensitive to heat-treatment and trypsin degradation. The present results, taken together with previous studies, indicate that the site of action of the inhibitor is not on the initiation phase of protein synthesis but rather at a step after the binding of aminoacyl-tRNA to ribosomes. The increased inhibitor activity found in dystrophic muscle is discussed.     

Interaction of the low molecular weight form of elongation factor 1 with guanine nucleotides and aminoacyl-tRNA.

A low molecular weight form of the eukaryotic polypeptide chain elongation factor 1 (EF-1α) has been extensively purified from pig liver to give an apparently homogeneous preparation, which seemed to be analogous to the bacterial elongation factor, EF-Tu (Iwasaki, K., Nagata, S., Mizumoto, K., and Kaziro, Y. (1974) J. Biol. Chem. 249, 5008). Thus, the interaction of the purified EF-1α with guanine nucleotides as well as aminoacyl-tRNA has been investigated and the following results have been obtained. (1) EF-1α when kept in the absence of glycerol lost its activity to promote the binding of aminoacylt-RNA to ribosomes though it retained the ability to bind guanine nucleotides. However, the former activity could be stabilized by the addition of 25% ($\text{v}\text{v}$) glycerol to the solution. (2) EF-1α formed a binary complex with guanine nucleotides such as GTP, GDP, 5′-guanylyl methylenediphosphonate or 5′-guanylyl imidodiphosphate. The molar ratio of EF-1α to GTP or GDP in the binary complex was shown to be 1. (3) The presence of a ternary complex containing EF-1α, GTP and aminoacyl-tRNA was demonstrated by several methods, i.e., (i) an increased heat stability of EF-1α in the presence of GTP and Phe-tRNA, (ii) a decrease in the amount of the EF-1α·GTP complex in the presence of aminoacyl-tRNA, (iii) a protection of the ester linkage of Phe-tRNA from hydrolysis at alkaline pH by the presence of both EF-1α and GTP, and (iv) the isolation of the complex by gel filtration.     

Mutually exclusive binding of messenger RNA and initiator methionyl transfer RNA to eukaryotic initiation factor 2

Eukaryotic initiation factor 2 (eIF-2), purified to at least 98% homogeneity as judged by polyacrylamide gel electrophoresis in sodium dodecyl sulfate, and containing no detectable amounts of eukaryotic initiation factor 4B (eIF-4B), is active both in the binding of Met-tRNA and in the binding of globin mRNA. The mRNA-binding activity is completely sensitive to competitive inhibition by Met-tRNA, provided GTP is present, but not by uncharged tRNA. By contrast, binding of mRNA to partially purified eIF-4B is not inhibited by Met-tRNA_f. These results establish that the only mRNA-binding component in the eIF-2 preparation is eIF-2 itself, and show that a given molecule of eIF-2 can either bind to a molecule of mRNA, or form a ternary complex with Met-tRNA_f and GTP, but cannot do both at once.     

Structural and functional properties of thesaurin a (42Sp50), the major protein of the 42 S particles present in Xenopus laevis previtellogenic oocytes

Thesaurin a is one of two protein components of a 42 S ribonucleoprotein particle that is very abundant in previtellogenic oocytes of Xenopus laevis. The primary function of the 42 S particle is the long-term storage of 5 S RNA and aminoacyl-tRNA. Thesaurin a is homologous to eukaryotic elongation factor 1 alpha (EF-1 alpha) and to prokaryotic elongation factor Tu (EF-Tu). Sequence comparison with EF-1 alpha and EF-Tu of different species indicates that thesaurin a is rather distantly related to all eukaryotic elongation factors. In spite of this, the secondary structure of thesaurin a, deduced from hydrophobic cluster analysis, is remarkably similar to that of EF-1 alpha and EF-Tu. The binding and catalytic properties of thesaurin a are also similar but not identical to those of EF-1 alpha. Like EF-1 alpha, purified thesaurin a binds tRNA, GDP, and GTP. Unlike EF-1 alpha, thesaurin a binds discharged tRNA more tightly than charged tRNA, and GTP more tightly than GDP. Thesaurin a also hydrolyzes GTP and catalyzes the mRNA-dependent binding of aminoacyl-tRNA to 80 S ribosomes. The functional properties of the 42 S particle are in general agreement with those of purified thesaurin a. In particular, the 42 S particle contains GTP and efficiently transfers aminoacyl-tRNA to 80 S ribosomes without addition of exogenous elongation factor.     

Comparison of cytoplasmic informosome proteins with RNA-binding proteins and translation initiation factors from rabbit reticulocytes.

Using one-and two-dimensional electrophoresis, the free and polyribosomal informosome proteins and a preparation of total RNA-binding proteins from rabbit reticulocytes were compared. It was shown that the major proteins of free and polyribosomal informosomes are similar only to the minor components of RNA-binding proteins. On the other hand, the major RNA-binding proteins, two of which are elongation translation factors EF-1L and EF2, can be present in informosome preparations only as minor components. The major proteins of polyribosomal informosomes do not coincide in terms of electrophoretic mobility with initiation factors eIF-2, eIF-2A, eIF-3, eIF-4A and eIF-4B. The major proteins of free informosomes differ in their electrophoretic mobility from initiation factors eIF-2A, eIF-4A and eIF-4B as well as from the alpha- and beta-subunits of initiation factor eIF-2.     

Purification and properties of nuclear and cytoplasmic DNA polymerases from JLS-V9 cells.

Four DNA polymerases, two enzymes from the nucleus and two from the cytoplasm, were purified 2000- to 7000-fold from continuous mouse cell-line (JLS-V9), by sequential column chromatography. Each of these polymerases require all the deoxynucleoside-5′-triphosphates in order to synthesize DNA, using activated DNA as a primer-template, and can copy the ribonucleotide strand of hybrid templates, but their rate of efficiency varies. The molecular weights of these DNA polymerases range from 35,000 to 160,000, as estimated by Sephadex column chromatography. Three out of the four DNA polymerases are probably a single polypeptide chain, since they have a single major band in polyacrylamide gel electrophoresis as well as one enzymatically active peak in guanidine hydrochloride gel filtration. The highly purified preparation of the high molecular weight cytoplasmic DNA polymerase contains two major bands in sodium dodecyl sulfate polyacrylamide gel electrophoresis and two enzymatically active peaks in guanidine hydrochloride gel filtration.     

Purification and characterization of a ribosome dissociation factor (eukaryotic initiation factor 6) from wheat germ.

A wheat germ ribosome dissociation factor, eukaryotic initiation factor 6 (eIF-6), has been purified almost to homogeneity from the 25 to 40% ammonium sulfate fraction of the postribosomal supernatant. This dissociation factor is distinct from initiation factor eIF-3 and its chromatographic properties permit its separation from the known wheat germ initiation factors. Under certain conditions, eIF-6 stimulates the incorporation of amino acids into polypeptides in a partially fractionated wheat germ cell-free system. The eight-step purification procedure developed includes chromatography on DEAE-cellulose, phosphocellulose, Sephadex G-75, and hydroxyapatite and yields a dissociation factor more than 80% pure. The purified factor is composed of a single polypeptide chain with a molecular weight of approximately 23,000 as determined by gel filtration chromatography and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It is an acidic protein which is heat labile and is inactivated by treatment with N-ethylmaleimide. The dissociation factor is much more effective in preventing the reassociation of 40 S and 60 S ribosomal subunits than in directly dissociating 80 S ribosomes. Like Escherichia coli IF-3, about 10 pmol of the dissociation factor are required to dissociate 1 pmol of ribosomes.     

Isolation of Eukaryotic Initiation Factor 2 from Rat Brain

Eukaryotic initiation factor 2 (eIF-2) was isolated from salt-washed microsomes of 4-day-old rat brain which show a high rate of protein synthesis. A three-step purification scheme was employed, including heparin-Sepharose, phosphocellulose, and DEAE-cellulose column chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the isolated factor revealed three polypeptides with molecular weights of 43,000, 54,000, and 59,000 and 90% purity. The rat brain eIF-2 forms ternary complexes with [3H]methionyl-tRNAi and GTP. In terms of specific activity, the purification does not correspond to that revealed by electrophoretic analysis. During purification there is an apparent loss of additional factors that modulates the activity of eIF-2 and explains the high rate of activity of the crude fraction.     

Mechanism of interferon action. Purification and substrate specificities of the double-stranded RNA-dependent protein kinase from untreated and interferon-treated mouse fibroblasts

The double-stranded RNA (dsRNA)-dependent protein kinase which catalyzes the phosphorylation of ribosome-associated protein P1 and the alpha subunit of eukaryotic protein synthesis initiation factor 2 (eIF-2) was purified and characterized from mouse fibroblast L929 cells treated with either natural or recombinant interferon and from untreated cells. The dsRNA-dependent P1/eIF-2 alpha kinase was purified at least 1,500-fold from interferon-treated cells; the kinase activity that catalyzed the phosphorylation of eIF-2 alpha copurified with protein P1. The yield of P1/eIF-2 alpha protein kinase activity obtained following purification from cells treated with interferon was about 5-10 times greater than the yield from an equivalent number of untreated cells. The purified protein kinase remained dsRNA dependent. When P1 kinase was activated by dsRNA, a major phosphopeptide designated Xds was phosphorylated; Xds was not phosphorylated from P1 which had not been activated by dsRNA. The apparent native molecular weight of the purified mouse L929 dsRNA-dependent kinase as determined by sedimentation analysis was about 62,000, comparable to the molecular weight of 67,000 determined for denatured L929 phosphoprotein P1 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified protein kinase was highly selective for the alpha subunit of protein synthesis initiation factor eIF-2 and endogenous protein P1. Kinase activity was dependent upon Mg2+, and the Km for ATP was determined to be 5 X 10(-6) M. Histones (H1, H2A-B, H3, and H4) and protein synthesis initiation factors other than eIF-2 (eIF-3, eIF-4A, eIF-4B, and eIF-5) were not substrates or were very poor substrates for the purified dsRNA-dependent protein kinase. N-Ethylmaleimide, ethylenediaminetetraacetic acid, AMP, pyrophosphate, spermine, spermidine, and high concentrations of potassium inhibited both P1 and eIF-2 alpha phosphorylation by the purified kinase, whereas ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid and phenanthroline did not significantly affect the phosphorylation of either protein P1 or eIF-2 alpha.     

Protein synthesis in yeast. Purification of elongation factor 3 from temperature-sensitive mutant 13-06 of the yeast Saccharomyces cerevisiae

An altered form of the elongation factor 3 (EF-3) has been purified to near homogeneity from a thermolabile yeast mutant ts 13-06. The isolation procedure involved chromatography on DEAE-Sephadex, CM-Sepharose, and hydroxylapatite columns. The final purification of this protein was obtained by affinity chromatography on an ATP-Sepharose column. Because of the extreme lability of the mutant protein, the yield was very poor. Silver stain analysis of the sodium dodecyl sulfate electrophoretograms indicated that the affinity-purified protein was better than 90% pure. From the studies of the physical and biochemical properties, the following characteristics of the purified wild type and the mutant protein have been established. The two proteins were indistinguishable by their molecular weight, amino acid composition, and isoelectric point. Purified mutant EF-3 was rapidly inactivated between 37 and 39 degrees C. Under this condition, wild type EF-3 was completely stable. Ribosome-dependent GTPase and ATPase activities of the mutant EF-3 were heat sensitive; GTPase activity was more labile than the ATPase activity. Mutant EF-3, after exposure to a nonpermissive temperature, failed to stimulate binding of the ternary complex of EF-1 X GTP X aminoacyl-tRNA to ribosome. The wild type protein was fully active under this condition. Other biochemical and physical properties of these two proteins are under current investigation.     

Protein synthesis in brine shrimp embryos. Dormant and developing embryos of Artemia salina contain equivalent amounts of chain initiation factors 2.

Dormant and developing embryos of Artemia salina contain equivalent amounts of eIF-2, the eukaryotic initiation factor which forms a ternary complex with GTP and Met-tRNAf. The factor was purified from 0.5 M NH4Cl ribosomal washes by (NH4)2SO4 fractionation, followed by chromatography on heparin-Sepharose, DEAE-cellulose, hydroxyapatite and phosphocellulose. Purified preparations from dormant and developing embryos have similar specific activities and nucleotide requirements. The mobility of both proteins in dodecylsulfate gel electrophoresis is indistinguishable, and each contains three major polypeptide chains of molecular weight 52 000, 45 000 and 42 000. Both proteins are also immunologically identical, and each stimulates amino acid incorporation in a cell-free system of protein synthesis. The binding of [35S]Met-tRNAf to 40-S ribosomal subunits is catalyzed by eIF-2 isolated from dormant or developing embryos and is dependent upon GPT and AUG. Binding of [35S]Met-tRNAf to 40-S ribosomal subunits, and ternary complex formation with eIF-2, GTP, and [35S]Met-tRNAf is stimulated 2--3-fold by a factor present in the 0.5 M NH4Cl ribosomal wash and which elutes from DEAE-cellulose at 50 mM KCl. This protein does not exhibit GTP-dependent binding of [35S]Met-tRNAf. Binding of GDP and GTP was investigated with purified eIF-2 from developing embryos. The factor forms a binary complex with GDP or GTP, and eIF-2-bound [3H]GDP exchanges very slowly with free nucleotides. Our results suggest that eIF-2 does not limit resumption of embryo development following encystment, nor does it limit mRNA translation in extracts from dormant embryos.     

Purification and characterization of initiation factor IF-E2 from rabbit reticulocytes.

Initiation factor IF-E2 was isolated from rabbit reticulocytes and purified 120-fold to near homogeneity by ammonium sulfate fractionation, column chromatography on DEAE-cellulose and phosphocellulose, and, when suitable, by sucrose density gradient centrifugation. The factor is a complex protein containing three nonidentical polypeptides of molecular weight 57,000, 52,000, and 36,000. It behaves as a complex throughout its purification and during polyacrylamide gel electrophoresis in nondenaturing buffer but its thress components are readily separated by electrophoresis in denaturing buffers. None of its components corresponds to any of the polypeptides of the other initiation factors or to any proteins of ribosomes washed in buffers containing a high salf concentration. A stoichiometric ratio of 1:1:1 was determined for the three polypeptides; based on the assumption of one copy each per complex, the calculated factor molecular weight is 145,000, a value in agreement with the measured value of 160,000. Initiation factor IF-E2 was radioactively labeled in vitro by reductive alkylation or by phosphorylation with a protein kinase also isolated from rabbit reticulocytes. Neither procedure causes a measurable change in the ability of the factor to form a ternary complex with GTP and the initiator methionyl-tRNA. 5'-Guanylyl-methylenediphosphonate may substitute for GTP, but only at relatively high concentrations. The binding of labeled initiation factor IF-E2 and methionyl-tRNA to the 40 S ribosomal subunit was studied by sucrose density gradient centrifugation. Appreciable binding of the factor is seen only when all three components of the ternary complex are included in the reaction mixture. The binding of either the factor or methionyl-tRNA was not stimulated by the addition of globin messenger RNA and initiation factor IF-E3. It was shown that all three polypeptide components of initiation factor IF-E2 are bound to these nascent initiation complexes.     

Further characterization of eukaryotic initiation factor 5 from rabbit reticulocytes. Immunochemical characterization and phosphorylation by casein kinase II

Eukaryotic initiation factor (eIF)-5, isolated from rabbit reticulocyte lysates, is a monomeric protein of Mr = 58,000-62,000. Immunochemical methods were employed to identify eIF-5 in crude cell lysates. Antisera against purified denatured eIF-5 were prepared in rabbits and characterized by immunoblotting and immunoprecipitation techniques using native and denatured eIF-5 as antigens. Monospecific antibodies to denatured eIF-5 were affinity-purified using eIF-5 blotted onto aminophenylthioether paper. Rabbit reticulocytes, HeLa cells and mouse L cells were lysed directly into a denaturing buffer containing 3% sodium dodecyl sulfate. The denatured proteins were analyzed by polyacrylamide gel electrophoresis followed by immunoblotting with anti-eIF-5 antibodies. With each lysate, one major immunoreactive polypeptide was observed whose molecular weight corresponded to that of purified eIF-5 (Mr = 58,000-62,000). No degradation products or precursor forms of molecular weight higher than 62,000 were detected in any lysate. These results indicate that isolated eIF-5 is the same size as that found in crude lysates. Additional characterization of eIF-5 indicates that purified eIF-5 can be phosphorylated at serine residues in vitro by casein kinase II. Furthermore, in vitro phosphorylated eIF-5 retains full biological activity in catalyzing the joining of 60 S ribosomal subunits to a preformed 40 S ribosomal initiation complex to form an 80 S initiation complex. Based on its specific activity, we demonstrate that 1 pmol of rabbit reticulocyte eIF-5 mediates the formation of approximately 180 pmol of 80 S initiation complex under the conditions of in vitro initiation reactions.     

Purification and properties of eukaryotic initiation factor 2 and its ancillary protein factor (Co-eIF-2A) from yeast Saccharomyces cerevisiae

Two peptide chain initiation factor activities, eIF-2y and Co-eIF-2A20y, were purified from the high speed supernatant fraction of the yeast Saccharomyces cerevisiae and their properties were studied. 1) In sodium dodecyl sulfate-polyacrylamide gels, purified eIF-2y showed two major polypeptide bands corresponding to molecular weights of 54,000 and 36,000. The Mr 54,000 band was significantly more intense than the Mr 36,000 band, indicating the possible presence of two polypeptides of equal molecular weight in this band. The molecular weight of eIF-2y, determined using a density gradient centrifugation method, was approximately 140,000. 2) In sodium dodecyl sulfate-polyacrylamide gel, purified Co-eIF-2A20y showed a single polypeptide band corresponding to a molecular weight of 20,000. A similar molecular weight for Co-eIF-2A20y was also found using a density gradient centrifugation method. 3) In partial reactions, eIF-2y bound Met-tRNAf in the presence of Mg2+. The reaction required GTP. Co-eIF-2A20y stimulated Met-tRNAf binding to eIF-2y (2-3-fold) and also rendered the complex stable to 3 X 10(-5) M aurintricarboxylic acid. 4) This Co-eIF-2A20y activity was heat-labile and N-ethylmaleimide-insensitive. 5) Antibodies were prepared by injecting rabbits with homogeneous Co-eIF-2A20y. Such anti-Co-eIF-2A20y inhibited (60%) protein synthesis in a yeast cell-free protein synthesizing system and completely blocked Co-eIF-2A20y stimulation of Met-tRNAf. 40 S initiation complex formation. Protein synthesis inhibition by anti-Co-eIF-2A20y was almost completely reversed by preincubation of the antibodies specifically with homogeneous Co-eIF-2A20y.     

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.