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.     

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 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.     

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.     

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.     

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.     

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.     

Studies on the role of eukaryotic nucleotide exchange factor in polypeptide chain initiation

Interactions of eukaryotic 5-dimethylaminonaphthalene-1-sulfonyl-initiation factor 2 (eIF-2) from rabbit reticulocytes and the guanine nucleotide exchange factor ( GEF ), Met-tRNAf, GTP, and GDP were monitored by changes in fluorescence anisotropy and radioactive filtration assays. At 1 mM Mg2+, radioactive filtration assays demonstrate that GEF is necessary for nucleotide exchange. We did not observe a GDP dependence in the association reaction of eIF-2 X GEF for GDP concentrations from 0.01 to 20 microM. This is in disagreement with the model: eIF-2 X GDP + GEF in equilibrium eIF-2 X GEF + GDP. The addition of GTP caused a decrease in fluorescence anisotropy which is interpreted as a dissociation of eIF-2 X GEF . We propose an asymmetrical model of ternary complex (eIF-2 X GTP X Met-tRNAf) formation where 1) GDP does not displace GEF and 2) GTP replaces GEF and presumably GDP. For reticulocyte eIF-2, phosphorylation of the alpha subunit greatly inhibits protein synthesis. This inhibition derives neither from failure of GEF to bind to eIF-2(alpha P) nor from greatly enhanced binding of GEF . The inhibition results from the requirement of very high levels of GTP (100 microM) to dissociate the eIF-2(alpha P) X GEF complex.     

The isolation and characterization from rabbit reticulocytes of two forms of eukaryotic initiation factor 2 having different beta-polypeptides

We have isolated from the high salt wash of rabbit reticulocyte ribosomes two forms of the polypeptide chain initiation factor 2 (eIF-2) which differ with respect to their beta-subunit, GDP content, and sensitivity to Mg2+ in ternary (eIF-2 X GTP X Met-tRNAf) and binary (eIF-2 X GDP) complex formation. The form of eIF-2 eluting first from a cation exchange (Mono S, Pharmacia) column has a beta-subunit of lower molecular weight (eIF-2(beta L] and a more acidic pI value than the form eluting at a higher salt concentration (eIF-2(beta H]. These two forms of eIF-2 beta-polypeptides are also detected in reticulocyte lysates when the proteins are resolved by two-dimensional isoelectric focusing-dodecyl sulfate polyacrylamide gel electrophoresis followed by immunoblotting. The peptide mapping of the isolated beta-subunits after limited proteolysis by papain, pancreatic protease, alpha-chymotrypsin, or Staphylococcus aureus V8 protease further demonstrates that the two forms of beta-subunits are not the product of a non-specific proteolytic action that occurred during the purification procedure, but rather reflects the existence in vivo of both forms of eIF-2. The GDP content of eIF-2(beta L) and eIF-2(beta H) is approximately 0.85 and 0.22 mol of GDP/mol of eIF-2, respectively. The KD for GDP of eIF-2(beta L) was lower (2.2 X 10(-9) M) than that of eIF-2(beta H) (6.0 X 10(-8) M). In the presence of 1 mM Mg2+, the activities of eIF-2(beta L) and eIF-2(beta H) in forming a binary and a ternary complex are inhibited 90 and 25%, respectively. The extent of Mg2+ inhibition and its reversal by the guanine nucleotide exchange factor is directly proportional to the amount of GDP bound to eIF-2. No inhibition by Mg2+ is observed when eIF-2-bound GDP is removed by alkaline phosphatase. In the presence of the guanine nucleotide exchange factor, both forms of eIF-2 are equally active in ternary complex formation, and the complex formed is quantitatively transferred to 40 S ribosomal subunits.     

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.     

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

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

Preparation and properties of a Met-tRNAf binding factor from rat liver and rat hepatoma.

A Met-tRNAf binding factor (IF-2) from the microsomal fraction of rat liver and rat hepatoma ascites cells was partially purified by ammonium sulphate fractionation, DEAE-cellulose and phosphocellulose chromatography. The factor binds [3H]Met-tRNAf only in the presence of either GTP or GMPPCP. Maximal binding takes place at 37 degrees C and in the absence of Mg++. The factor is specific for Met-tRNAf and does not bind Phe-tRNA from rat liver or from E. coli. The ternary complex [Met-tRNAf . IF-2 . GTP1 binds to 40 S ribosomal subunits from rat liver in the absence of mRNA or poly(A, G, U) without GTP hydrolysis. GDP as well as aurintricarboxylic acid inhibit the ternary complex formation. Both factors are rapidly inactivated by N-ethylmaleimide treatment and by preincubation at 45 degrees C. Heat inactivation is partially prevented by GTP and GDP. With regard to the functional properties there are no significant differences between IF-2 from normal liver and hepatoma cells. On the other hand heat denaturation compared to the rat liver factor, which may be due to differences in contaminating proteins.     

Natural mRNA is required for directing Met-tRNA(f) binding to 40S ribosomal subunits in animal cells: involvement of Co-eIF-2A in natural mRNA-directed initiation complex formation

Two protein factors, eIF-2 as well as a high molecular weight protein complex from reticulocyte ribosomal high-salt wash which we term Co-eIF-2, promote Met-tRNA(f) binding to 40S ribosomes. This binding is dependent on the presence of an AUG codon or natural mRNAs [Roy et al. (1984) Biochem. Biophys. Res. Commun. 122, 1418-1425]. Co-eIF-2 contains two component activities, Co-eIF-2A and Co-eIF-2C. Previously, we have purified an 80-kDa polypeptide containing Co-eIF-2A activity and showed that this polypeptide is a component of Co-eIF-2 and is responsible for Co-eIF-2A activity in Co-eIF-2 [Chakravarty et al. (1985) J. Biol. Chem. 260, 6945-6949]. We now report purification of a protein complex (subunits of Mr 180K, 110K, 65K, 63K, 53K, 50K, 43K, and 40K) containing Co-eIF-2C activity and devoid of Co-eIF-2A activity. In SDS-PAGE, the purified Co-eIF-2C preparation and an eIF-3 preparation (purified in Dr. A. Wahba's laboratory) separated into seven similar major polypeptides (Mr 110K, 65K, 63K, 53K, 50K, 43K, and 40K). The 50-kDa polypeptide in Co-eIF-2C was immunoreactive with a monoclonal antibody against eIF-4A (50 kDa). We have studied the roles of purified Co-eIF-2A and Co-eIF-2C activities in ternary and Met-tRNA(f).40S ribosome complex formation. The results are as follows: (1) At low and presumably physiological factor concentration (30 nM), eIF-2 did not form detectable levels of ternary complex. Moreover, such complex formation was totally dependent on the presence of Co-eIF-2A and/or Co-eIF-2C.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Binding of ATP to eukaryotic initiation factor 2. Differential modulation of mRNA-binding activity and GTP-dependent binding of methionyl-tRNAMetf

Eukaryotic initiation factor 2 (eIF-2) is shown to bind ATP with high affinity. Binding of ATP to eIF-2 induces loss of the ability to form a ternary complex with Met-tRNAf and GTP, while still allowing, and even stimulating, the binding of mRNA. Ternary complex formation between eIF-2, GTP, and Met-tRNAf is inhibited effectively by ATP, but not by CTP or UTP. Hydrolysis of ATP is not required for inhibition, for adenyl-5'-yl imidodiphosphate (AMP-PNP), a nonhydrolyzable analogue of ATP, is as active an inhibitor; adenosine 5'-O-(thiotriphosphate) (ATP gamma S) inhibits far more weakly. Ternary complex formation is inhibited effectively by ATP, dATP, or ADP, but not by AMP and adenosine. Hence, the gamma-phosphate of ATP and its 3'-OH group are not required for inhibition, but the beta-phosphate is indispensible. Specific complex formation between ATP and eIF-2 is shown 1) by effective retention of Met-tRNAf- and mRNA-binding activities on ATP-agarose and by the ability of free ATP, but not GTP, CTP, or UTP, to effect elution of eIF-2 from this substrate; 2) by eIF-2-dependent retention of [alpha-32P]ATP or dATP on nitrocellulose filters and its inhibition by excess ATP, but not by GTP, CTP, or UTP. Upon elution from ATP-agarose by high salt concentrations, eIF-2 recovers its ability to form a ternary complex with Met-tRNAf and GTP. ATP-induced inhibition of ternary complex formation is relieved by excess Met-tRNAf, but not by excess GTP or guanyl-5'-yl imidodiphosphate (GMP-PNP). Thus, ATP does not act by inhibiting binding of GTP to eIF-2. Instead, ATP causes Met-tRNAf in ternary complex to dissociate from eIF-2. Conversely, affinity of eIF-2 for ATP is high in the absence of GTP and Met-tRNAf (Kd less than or equal to 10(-12) M), but decreases greatly in conditions of ternary complex formation. These results support the concept that eIF-2 assumes distinct conformations for ternary complex formation and for binding of mRNA, and that these are affected differently by ATP. Interaction of ATP with an eIF-2 molecule in ternary complex with Met-tRNAf and GTP promotes displacement of Met-tRNAf from eIF-2, inducing a state favorable for binding of mRNA. ATP may thus regulate the dual binding activities of eIF-2 during initiation of translation.     

Affinity labeling by a photoreactive GTP analogue of the alpha-subunit of eukaryotic initiation factor eIF-2 in different initiation complexes

The interaction of GTP with initiation factor eIF-2 in different complexes was studied by affinity labeling using a derivative of [3H]GTP carrying a photoreactive group in the alpha-phosphate moiety. In the binary complex [eIF-2.GTP analogue], in the ternary complex [eIF-2.GTP analogue.Met-tRNAf] as well as in the eIF-2. eIF-2B complex the alpha-subunit of eIF-2 was found to be specifically labeled. GTP is concluded to interact during polypeptide chain initiation with the alpha-subunit of eIF-2 at least by its alpha-phosphate group.     

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).     

Protein synthesis in rabbit reticulocytes: requirements for Met-tRNAf . 40S preinitiation complex formation with AUG-codon and physiological mRNAs

Under standard conditions, in the presence of GTP, highly purified eIF-2 and Co-eIF-2 factor preparations efficiently stimulated AUG-codon dependent but not physiological mRNA-dependent Met-tRNAf binding to 40S ribosomes. Replacement of GTP by a nonhydrolyzable GTP analog, GMP-PNP, in the above system, gave significant stimulation of Met-tRNAf binding to 40S ribosomes dependent on physiological mRNAs. Lower but significant stimulation of Met-tRNAf binding to 40S ribosomes was also observed when GTP was used in the presence of nucleoside 5'-diphosphate kinase (NDK) and ATP. ATP alone in the absence of NDK had no significant effect. This is the first report on the formation of a stable Met-tRNAf . 40S initiation complex dependent on physiological mRNAs and the factor requirements for such complex formation.     

Evaluation and significance of kinetic parameters governing function of protein synthesis initiation factors eIF-2 and eIF-2B

Published data dealing with the formation of the ternary complex eIF-2 X GTP X met-tRNAi involved in eukaryotic initiation have been evaluated to calculate the expected inhibition by GDP and the role of eIF-2B in limiting this inhibition. It is concluded that cellular levels of GDP are unlikely seriously to inhibit ternary complex formation if the reaction can proceed to equilibrium. However, derivation of 'on' and 'off' rates for the interaction of GTP and GDP with eIF-2 demonstrates that these are too slow in the absence of eIF-2B to support active protein synthesis, particularly if eIF-2 is released from ribosomes as eIF-2 X GDP. Whilst eIF-2 X GDP and eIF-2 X GTP appear to dissociate equally slowly, it is concluded that GDP binds to eIF-2 100-times faster than GTP. Addition of eIF-2B has the effect of raising k-1 for both GDP and GTP several hundred-fold and k+1 50- and 7000-fold, respectively. Thus, a kinetic block can be relieved even if there is no change in the thermodynamic state. Phosphorylation of the alpha-subunit of eIF-2 appears to affect only those parameters influenced by eIF-2B. The reported rescue of inhibited lysates by addition of 1 mM GTP is not by mass action but by some other mechanism. Consideration of the kinetic parameters favours the formation of a ternary complex of eIF-2 X eIF-2B X GDP en route to eIF-2 X GTP as opposed to displacement of GDP from eIF-2 X GDP by eIF-2B.     

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.