Protein synthesis in rabbit reticulocytes. Purification and properties of an Mr 80,000 polypeptide (Co-eIF-2A80) with Co-eIF-2A activity

The high molecular weight protein complex, Co-eIF-2, contains both Co-eIF-2A and Co-eIF-2C activities (Bagchi, M. K., Banerjee, A. C., Roy, R., Chakravarty, I., and Gupta, N. K. (1982) Nucleic Acids Res. 10, 6501-6510). Co-eIF-2A stimulated Met-tRNAf binding to eukaryotic initiation factor-2 (eIF-2) both in the presence and absence of Mg2+. Co-eIF-2C stimulates Met-tRNAf binding to eIF-2 in the presence of Mg2+ by relieving Mg2+ inhibition of ternary complex formation from eIF-2. Co-eIF-2 protein complex contains several polypeptides including Mr 80,000 and 50,000 polypeptides. Three polypeptides (Mr 80,000, 50,000 and 25,000) are present in 0.5 M KCl ribosomal salt wash and each possesses Co-eIF-2A activity. Mr 80,000 polypeptide (Co-eIF-2A80) has been purified to homogeneity and its properties studied. 1) Co-eIF-2A80 stimulated Met-tRNAf binding to eIF-2 and the complex formed was resistant to aurintricarboxylic acid. 2) Co-eIF-2A80 activity was N-ethylmaleimide-resistant and heat-labile; it was destroyed by heating at 55 degrees C for 4 min. 3) Antibodies prepared against homogeneous Co-eIF-2A80 strongly inhibited protein synthesis in reticulocyte lysates and, also, eIF-2 and Co-eIF-2 promoted Met-tRNAf binding to 40 S ribosomes. Inhibition of protein synthesis in reticulocyte lysates was overcome by preincubation of anti-Co-eIF-2A80 with homogeneous Co-eIF-2A80 and was partially overcome by similar preincubation with Co-eIF-2. 4) Upon limited digestion with Staphylococcus aureus V8 protease, the homogeneous Co-eIF-2A80 gave two major polypeptide fragments (Mr 50,000 and 25,000). Upon similar treatment, an Mr 80,000 polypeptide band isolated from the sodium dodecyl sulfate-gel of the Co-eIF-2 protein complex gave four major polypeptide fragments, and two of these fragments (Mr 50,000 and 25,000) were similar to those given by Co-eIF-2A80, indicating that this Mr 80,000 polypeptide band contains the Co-eIF-2A80 component. We suggest that Co-eIF-2A80 is a component of Co-eIF-2 and is also essential for Co-eIF-2 activity and overall peptide chain initiation.     

Protein synthesis in rabbit reticulocytes. A study of the mechanism of Co-eIF-2 action

The characteristics of component activities in Co-eIF-2 (where eIF is eukaryotic initiation factor) protein complex have been studied. (i) At limiting concentrations, Co-eIF-2 promoted rapid GDP binding to eIF-2 and also GDP displacement from eIF-2 X GDP during ternary complex formation in the presence of GTP and Mg2+ (Co-eIF-2C activity) but did not significantly stimulate ternary complex formation by eIF-2. (ii) At higher concentrations, Co-eIF-2 significantly enhanced ternary complex formation by eIF-2 and also rendered the complex stable to aurintricarboxylic acid presumably as Co-eIF-2 became physically bound to the ternary complex (Co-eIF-2A activity). (iii) Ternary complex preformed in the presence of Co-eIF-2 and without Mg2+ dissociated upon subsequent addition of Mg2+ (Co-eIF-2B activity). This dissociation reaction was presumably due to loss of interaction of the Co-eIF-2A component in Co-eIF-2 with the ternary complex (reversal of Co-eIF-2A activity) as the complex became increasingly sensitive to aurintricarboxylic acid with increasing Mg2+ concentration. In another study, purified eIF-2 was freed of bound GDP by treatment with alkaline phosphatase and the characteristics of native and GDP-free eIF-2 were compared. (i) One mM Mg2+ inhibited (60%) ternary complex formation by native eIF-2 but not by GDP-free eIF-2. Addition of exogenous GDP rendered GDP-free eIF-2 sensitive to Mg2+ indicating that Mg2+ inhibition is due to eIF-2-bound GDP. (ii) In the presence of Mg2+, Co-eIF-2 stimulated similarly ternary and Met-tRNAf X 40 S X AUG complex formation by both native and GDP-free eIF-2. Such stimulatory activity in each case was strongly inhibited by prior phosphorylation of eIF-2 alpha subunit by heme-regulated translational inhibitor. (iii) Ternary complexes preformed using either native and GDP-free eIF-2 and excess Co-eIF-2A80 in the absence of Mg2+ did not form Met-tRNAf X 40 S X AUG complex. They required trace amounts of Co-eIF-2 for such activity.     

Protein synthesis in rabbit reticulocytes. A study of the roles of Co-eIF-2, Co-eIF-2A80, and GDP in peptide chain initiation

The roles of Co-eIF-2, Co-eIF-2A80, and GDP in ternary complex and Met-tRNAf X 40 S initiation complex formation were studied. 1) Partially purified eukaryotic initiation factor 2 (eIF-2) (50% pure) preparations contained 0.4-0.6 pmol of bound GDP/pmol of eIF-2. eIF-2 purity was calculated from ternary complex formation in the absence of Mg2+ and in the presence of excess Co-eIF-2. 2) In the absence of Mg2+, approximately 30% of the potentially active eIF-2 molecules formed ternary complexes, and both Co-eIF-2 and Co-eIF-2A80 were equally effective in full activation of the eIF-2 molecules for ternary complex formation. 3) In the presence of Mg2+, approximately 10% of the potentially active eIF-2 molecules formed ternary complexes in the absence of ancillary factors, and the ancillary factors Co-eIF-2A80 and Co-eIF-2 raised the incorporation to 20 and 50% of the eIF-2 molecules, respectively. 4) In the absence of Mg2+, [3H]GDP in preformed eIF-2 X [3H]GDP was readily displaced by GTP during ternary complex formation. 5) In the presence of Mg2+, [3H]GDP remained tightly bound to eIF-2 and ternary complex formation was inhibited. Co-eIF-2, but not Co-eIF-2A80, was effective in promoting [3H]GDP displacement and the former was more effective in promoting ternary complex formation than the latter. 6) eIF-2 X [3H]GDP was converted to eIF-2 X [3H] GTP by incubation in the presence of nucleoside-5'-diphosphate kinase and ATP, but the eIF-2 X [3H]GTP thus formed did not bind Met-tRNAf in the presence of Mg2+ and required exogeneous addition of Co-eIF-2 and GTP for ternary complex formation and GTP displacement. 7) In the absence of Mg2+, the increased ternary complex formed in the presence of eIF-2 X [3H] GDP and Co-eIF-2A80 (with accompanying loss of [3H] GDP) was inactive in a subsequent reaction, which involves Met-tRNAf transfer to 40 S ribosomes (in the presence of Mg2+), and required trace amounts of Co-eIF-2 for such activity. Based on the above observations, we have suggested a two-step activation of eIF-2 molecules by the Co-eIF-2 protein complex for functional ternary complex formation. One of these steps involves the Co-eIF-2A component of Co-eIF-2. This activation results in stimulated Met-tRNAf binding to eIF-2 and is most apparent in the absence of Mg2+ and with aged eIF-2 molecules.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

A comparative study of the characteristics of eIF-2 and eIF-2-ancillary factor activities from yeast Saccharomyces cerevisiae and rabbit reticulocytes

The characteristics of yeast eukaryotic initiation factor 2 (eIF-2) and Co-eIF-2A have been studied and compared with those of the corresponding factors from rabbit reticulocytes. 1) Unlike eIF-2r, purified eIF-2y did not contain bound GDP. 2) Purified eIF-2y preparation contained GTPase activity and dephosphorylated GTP to GDP. 3) An anti-eIF-2r preparation which predominantly precipitated the gamma-subunit (Mr 54,000) of eIF-2r also precipitated the larger subunit (Mr 54,000) of eIF-2y. 4) Unlike eIF-2r, ternary complex formation by eIF-2y was not inhibited by Mg2+. 5) Both Co-eIF-2A20y and Co-eIF-2r significantly enhanced Met-tRNAf binding to eIF-2y and, again, Mg2+ did not have any effect on this stimulated Met-tRNAf binding to eIF-2y. 6) Both Co-eIF-2A20y and Co-eIF-2r were similarly effective in stimulating Met-tRNAf binding to eIF-2r in the absence of Mg2+. However, in the presence of Mg2+, Co-eIF-2A20y was significantly less effective than Co-eIF-2r as Co-eIF-2A20y did not promote displacement of GDP from eIF-2r X GDP. 7) eIF-2y bound [3H]GDP and this binding was significantly enhanced in the presence of Mg2+. Also, [3H]GDP in the preformed eIF-2y X [3H]GDP complex was rapidly exchanged with exogenously added unlabeled GDP in the presence of Mg2+. Co-eIF-2A20y had no effect on GDP binding to eIF-2y nor on GDP exchange reactions. 8) Reticulocyte heme-regulated protein synthesis inhibitor, which phosphorylated almost completely (in excess of 80%) the alpha-subunit (Mr 38,000) of eIF-2r, also phosphorylated similarly the smaller subunit (Mr 36,000) of eIF-2y. However, such phosphorylation had no significant effect on ternary complex formation, GDP binding, and GDP exchange reactions.     

Protein synthesis in yeast Saccharomyces cerevisiae. Purification of Co-eIF-2A and 'mRNA-binding factor(s)' and studies of their roles in Met-tRNAf.40S.mRNA complex formation

Antibodies prepared against a homogeneous preparation of Co-eIF-2A20 [Ahmad et al. (1985) J. Biol. Chem. 260, 6955-6959] reacted with several polypeptides including an 80-kDa polypeptide present in a crude yeast ribosomal salt wash. This 80-kDa polypeptide, containing Co-eIF-2A (Co-eIF-2A80) activity, has been extensively purified using a two-step purification procedure involving an immunoaffinity column chromatograph prepared using antibodies against Co-eIF-2A20 (fraction II) and hydroxyapatite chromatography (fraction III). The factors, eIF-2 + homogeneous Co-eIF-2A80 (fraction III) promoted Met-tRNAf.40S complex formation with an AUG codon but not with a physiological mRNA or a polyribonucleotide messenger poly(U,G) whereas eIF-2 + a partially purified Co-eIF-2A80 preparation (fraction II) promoted Met-tRNAf.40S complex formation with an AUG codon as well as with globin mRNA and poly(U,G) messenger. This factor-promoted Met-tRNAf binding to 40S ribosomes depends absolutely on the presence of a polyribonucleotide messenger containing an initiation codon (such as AUG or GUG). Other polyribonucleotide messengers tested, such as poly(U), poly(A) and poly(A,C) were completely ineffective in this binding reaction. This result indicates that the Met-tRNAf.40S.mRNA complex is formed by a direct interaction between Met-tRNAf, 40S ribosomes and the initiation site in mRNA. A mechanism has been proposed for Met-tRNAf.40S.mRNA complex formation in yeast.     

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.     

Protein synthesis in rabbit reticulocytes: characteristics of CO-eIF-2 protein complex.

A high molecular weight reticulocyte protein factor, named Co-eIF-2, contains Co-eIF-2A, Co-eIF-2B, and Co-eIF-2C activities and stimulates Met-tRNAf binding to eIF-2 both in the presence and absence of Mg2+. Some characteristics of this stimulation in the absence of Mg2+ are: (1) Stimulation is most pronounced at low eIF-2 levels. (2) Stimulation is partially resistant to heat and NEM treatment, and thus appears to be due to the combined action of both heat and NEM-insensitive Co-eIF-2A, and heat and NEM-sensitive Co-eIF-2C activities. (3) [3H]GDP bound in eIF-2 . [3H]GDP complex is rapidly displaced by unlabelled GTP during ternary complex formation Co-eIF-2 stimulates Met-tRNAf binding to eIF-2 even when added after the [3H]GDP from eIF-2 . [3H]GDP has been completely displaced. This indicates that Co-eIF-2-stimulation is not due to GDP displacement from eIF-2 . GDP. We propose that eIF-2 molecules become inactive in the presence of Mg2+ and at high dilution, and Co-eIF-2 restores the inactive eIF-2 molecules into an active form.     

Binding of ATP and messenger RNA by the beta-subunit of eukaryotic initiation factor 2.

In addition to forming a ternary complex with Met-tRNA(f) and GTP, eukaryotic initiation factor 2 (eIF-2) recognizes a specific site in mRNA molecules. Both binding activities are regulated by ATP, which itself binds tightly and specifically to eIF-2. Denaturation of eIF-2 with urea leads to complete loss of Met-tRNA(f) binding activity, while mRNA binding activity is stable. Hence, distinct conformational features in eIF-2 are required for ternary complex formation and for binding of mRNA. Chromatography of eIF-2 over ATP-agarose, in denaturing conditions that induce polypeptide subunit dissociation, results in selective retention of the beta-subunit of eIF-2. Isolated beta-subunit is capable of binding mRNA as well as ATP. Cibacron blue 3G-A binds tightly to eIF-2 and inhibits the binding of mRNA. This inhibition is relieved upon addition of ATP, showing that Cibacron blue 3G-A competes with ATP for eIF-2. eIF-2 beta subunit, active in binding of mRNA, is recovered upon chromatography of eIF-2 in denaturing conditions over matrix-bound Cibacron blue 3G-A. These results show that the ability of eIF-2 to bind mRNA and its ability to bind ATP are both lodged within remarkably stable domains of its beta-subunit. During initiation of protein synthesis, the eIF-2 beta subunit may thus interact with three ligands important for translational control: Met-tRNA(f), mRNA and ATP.     

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.     

Purification and characterization of sea urchin initiation factor 2. The requirement of guanine nucleotide exchange factor for the release of eukaryotic polypeptide chain initiation factor 2-bound GDP

Protein synthesis in sea urchin eggs is stimulated dramatically upon fertilization. We previously demonstrated that this stimulation is primarily due to an increase in the rate of polypeptide chain initiation which in turn may be regulated at the level of recycling of eukaryotic initiation factor 2 (eIF-2) (Colin, A. M., Brown, B. D., Dholakia, J. N., Woodley, C. L., Wahba, A. J., and Hille, M. B. (1987) Dev. Biol. 123, 354-363). We have now purified eIF-2 from sea urchin Strongylocentrotus purpuratus blastulae to apparent homogeneity by chromatography on DEAE-cellulose, phosphocellulose, Mono Q, Mono P, and Mono S columns. The factor, which differs from mammalian eIF-2, is composed of three non-identical subunits with apparent molecular weights of 40,000-alpha; 47,000-beta, and 58,000-gamma as estimated by sodium dodecyl-polyacrylamide gel electrophoresis. Antibodies raised against rabbit reticulocyte eIF-2 do not cross-react with sea urchin eIF-2. The binding of Met-tRNA(f) to sea urchin eIF-2 is totally dependent on GTP. A 4-fold stimulation in the rate of protein synthesis in unfertilized sea urchin egg extracts is observed by the addition of 1 micrograms of purified eIF-2. The factor also binds GDP to form a binary (eIF-2.GDP) complex which is stable in the presence of Mg2+. GDP binding to sea urchin eIF-2 inhibits ternary (eIF-2-GTP.[35S]Met-tRNA(f) complex formation. The rabbit reticulocyte guanine nucleotide exchange factor (GEF) catalyzes the exchange of GDP bound to sea urchin eIF-2 for GTP and stimulates ternary complex formation. The requirement of GEF for the recycling of eIF-2 suggests that protein synthesis in sea urchins is similar to that in mammalian systems and may also be regulated at the level of GEF activity. The reticulocyte heme-controlled repressor phosphorylates the alpha-subunit of eIF-2 from both sea urchins and rabbit reticulocytes. However, casein kinase II which phosphorylates the beta-subunit of the reticulocyte factor specifically phosphorylates the alpha-subunit of sea urchin eIF-2. In this respect, the sea urchin factor is similar to eIF-2 isolated from other nonmammalian sources. Since both heme controlled repressor and casein kinase II phosphorylate the alpha-subunit of sea urchin eIF-2 caution should be exercised when interpreting the significance of eIF-2(alpha) phosphorylation in sea urchins.     

The effect of Mg2+ and guanine nucleotide exchange factor on the binding of guanine nucleotides to eukaryotic initiation factor 2

A major site of regulation of polypeptide chain initiation is the binding of Met-tRNA to 40 S ribosomal subunits which is mediated by eukaryotic initiation factor 2 (eIF-2). The formation of ternary complex, eIF-2.GTP.Met-tRNA, is potently inhibited by GDP. Measurement of the parameters for guanine nucleotide binding to eIF-2 is critical to understanding the control of protein synthesis by fluctuations in cellular energy levels. We have compared the dissociation constants (Kd) of eIF-2.GDP and eIF-2.GTP and find that GDP has a 400-fold higher affinity for GDP than GTP. The Kd for GDP is almost an order of magnitude less than has been reported previously. The difference between the Kd values for the two nucleotides is the result of a faster rate constant for GTP release, the rate constants for binding being approximately equal. This combination of rate constants and low levels of contaminating GDP in preparations of GTP can explain the apparently unstable nature of eIF-2.GTP observed by others. Mg2+ stabilizes binary complexes slowing the rates of release of nucleotide from both eIF-2.GDP and eIF-2.GTP. The competition between GTP and GDP for binding to eIF-2.guanine nucleotide exchange factor complex has been measured. A 10-fold higher GTP concentration than GDP is required to reduce [32P] GDP binding to eIF-2.guanine nucleotide exchange factor complex by 50%. The relevance of this competition to the regulation of protein synthesis by energy levels is discussed.     

Factors from wheat germ that enhance the activity of eukaryotic initiation factor eIF-2. Isolation and characterization of Co-eIF-2 beta

A factor has been isolated from wheat germ that enhances the ability of initiation factor 2 (eIF-2) to form a ternary complex with GTP and Met-tRNAf and enhances the binding of Met-tRNAf to 40 s ribosomal subunits. This factor, designated Co-eIF2 beta, is a monomeric protein with a molecular weight of approximately 83,000. Wheat germ eIF-2 forms a stable binary complex with GDP but not with GTP. Co-eIF-2 beta enhances the formation of an eIF-2 . GDP complex, but does not enable eIF-2 to form a stable complex with GTP.     

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.     

Protein synthesis in rabbit reticulocytes: Mg2+-inhibition of ternary complex (Met-tRNA(f).eIF-2.GTP) formation by reticulocyte eIF-2

There are conflicting reports regarding Mg2+-inhibition of ternary complex formation by reticulocyte eIF-2. Several laboratories have reported that eIF-2 is isolated as eIF-2.GDP and Mg2+ inhibits ternary complex formation, as in the presence of Mg2+, GDP remains tightly bound to eIF-2 and prevents ternary complex formation. A protein factor, GEF is necessary for GDP displacement and subsequent ternary complex formation. Other laboratories have reported that Mg2+ has no effect on eIF-2 activity and eIF-2 forms near stoichiometric amount of ternary complex in the presence of Mg2+. In this paper, we provide evidence which suggests that the Mg2+-insensitive eIF-2 activity as reported by several laboratories might have been the result of the use of high Met-tRNA(f) concentrations in their assays as the nucleotides in excess tRNA bound Mg2+ in the reaction mixture and there was no free Mg2+ available to inhibit eIF-2 activity. Our data will show that the addition of excess tRNA promotes non-enzymatic GDP displacement from eIF-2.GDP and relieves Mg2+ inhibition.     

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.     

Regulation of protein synthesis in eukaryotes. Mode of action of eRF, an eIF-2-recycling factor from rabbit reticulocytes involved in GDP/GTP exchange

The rate of initiation of protein synthesis appears to be controlled at the level of recycling of eIF-2. In this process a new factor, designated eRF, plays an important role. The factor has been purified from the post-ribosomal supernatant and has been called formerly anti-HRI and anti-inhibitor [Amesz, H., Goumans, H., Haubrich-Morree, Th., Voorma, H.O., and Benne, R. (1979) Eur. J. Biochem. 98, 513-520]. Its effect on the initiation of protein synthesis has been studied in several assays: a small but distinct effect is found in the assay for the formation of a ternary complex between eIF-2, GTP and Met-tRNA; a 4-5-fold stimulation is obtained in assays for 40S preinitiation complex formation and in the methionyl-puromycin reaction. In the latter assay a catalytic use of eIF-2 occurs provided that eRF is present. eRF forms a complex with eIF-2 which results in a decrease of the affinity of eIF-2 for GDP, giving it the properties of a GDP/GTP exchange factor. The model stresses the catalytic use of eIF-2 in initiation provided that conditions are met for GDP/GTP exchange by a transient complex formation between eIF-2 and eRF. On the other hand, it is shown that phosphorylation of eIF-2 by the hemin-regulated inhibitor (HRI) abolishes the recycling of eIF-2, by the formation of another stable complex comprising eIF-2 alpha P, GDP and eRF.     

Photoaffinity labeling of the cap-binding protein complex with ATP/dATP. Differential labeling of free eukaryotic initiation factor 4A and the eukaryotic initiation factor 4A component of the cap-binding protein complex with [alpha-32P]ATP/dATP

It has been suggested that the cap-binding protein complex is involved in ATP-mediated melting of 5'-mRNA secondary structure to facilitate ribosome binding during initiation of translation in eukaryotic cells (Edery, I., Lee, K. A. W., and Sonenberg, N. (1984) Biochemistry 23, 2456-2462). Consequently, we have studied the interaction of dATP/ATP with the eukaryotic cap-binding protein complex by UV photoaffinity labeling. UV irradiation of the cap-binding protein complex in the presence of [alpha-32P]dATP/ATP resulted in the cross-linking of this compound to the 50-kDa polypeptide of the complex. This polypeptide is almost identical to the previously characterized eukaryotic initiation factor (eIF) 4A. We examined the ability of dATP/ATP to cross-link to eIF-4A and found that it cross-links less efficiently (approximately 60-fold on a molar basis) compared to the cross-linking obtained for the eIF-4A component of the cap-binding protein complex. Irradiation of purified eIF-4A together with the cap-binding protein complex in the presence of [alpha-32P]dATP resulted in greater than additive labeling of the eIF-4A component of the cap-binding protein complex and purified eIF-4A, suggesting a synergistic interaction between purified eIF-4A, the cap-binding protein complex, and dATP/ATP. We also report that photoaffinity labeling of eIF-4A and the eIF-4A component in the cap-binding protein complex is stimulated by eIF-4B, but not by other initiation factors or mRNA.     

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.     

Cap binding protein complex that restores protein synthesis in heat-shocked Ehrlich cell lysates contains highly phosphorylated eIF-4E

Cell-free protein-synthesizing systems derived from Ehrlich ascites tumor cells that have been exposed to elevated temperatures retain the inhibition of translation that is seen at the cellular level. A multisubunit cap binding protein complex able to restore protein synthesis in these cell free systems was purified from Ehrlich ascites tumor cells via affinity chromatography using m7GTP-Sepharose and fast protein liquid chromatography on Mono Q. The purified complex contains an Mr 220,000 polypeptide (p220) and an Mr 28,000 polypeptide (p28), both of which are components of eukaryotic initiation factor 4F (eIF-4F). p28 is identical to eIF-4E. Restoring activity was relatively free of the Mr 46,000 polypeptide (p46) that is the third component of eIF-4F and does not appear to be dependent on its presence. p28 associated in a complex with p220 is 85% phosphorylated; however, the majority of p28 is not associated with p220, and this free form is only about 50% phosphorylated. The correlation between association of p28 with p220 and high levels of p28 phosphorylation suggests a possible role for phosphorylation in association of p220 with p28.