Archaeal translation initiation revisited: the initiation factor 2 and eukaryotic initiation factor 2B alpha-beta-delta subunit families

As the amount of available sequence data increases, it becomes apparent that our understanding of translation initiation is far from comprehensive and that prior conclusions concerning the origin of the process are wrong. Contrary to earlier conclusions, key elements of translation initiation originated at the Universal Ancestor stage, for homologous counterparts exist in all three primary taxa. Herein, we explore the evolutionary relationships among the components of bacterial initiation factor 2 (IF-2) and eukaryotic IF-2 (eIF-2)/eIF-2B, i.e., the initiation factors involved in introducing the initiator tRNA into the translation mechanism and performing the first step in the peptide chain elongation cycle. All Archaea appear to posses a fully functional eIF-2 molecule, but they lack the associated GTP recycling function, eIF-2B (a five-subunit molecule). Yet, the Archaea do posses members of the gene family defined by the (related) eIF-2B subunits alpha, beta, and delta, although these are not specifically related to any of the three eukaryotic subunits. Additional members of this family also occur in some (but by no means all) Bacteria and even in some eukaryotes. The functional significance of the other members of this family is unclear and requires experimental resolution. Similarly, the occurrence of bacterial IF-2-like molecules in all Archaea and in some eukaryotes further complicates the picture of translation initiation. Overall, these data lend further support to the suggestion that the rudiments of translation initiation were present at the Universal Ancestor stage.     

A eukaryotic translation initiation factor 2-associated 67 kDa glycoprotein partially reverses protein synthesis inhibition by activated double-stranded RNA-dependent protein kinase in intact cells

A eukaryotic translation initiation factor 2 (eIF-2)-associated 67 kDa glycoprotein (p67) protects the eIF-2 alpha-subunit from inhibitory phosphorylation by eIF-2 kinases, and this promotes protein synthesis in the presence of active eIF-2 alpha kinases in vitro [Ray, M. K., et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 539-543]. We have now examined the effect of overexpression of this cellular eIF-2 kinase inhibitor in an in vivo system using transiently transfected COS-l cells. In this system, coexpression of genes that inhibit PKR activity restores translation of plasmid-derived mRNA. We now report the following. (1) Transient transfection of COS-1 cells with a p67 expression vector increased p67 synthesis by 20-fold over endogenous levels in the isolated subpopulation of transfected cells. (2) Cotransfection of p67 cDNA increased translation of plasmid-derived mRNAs. (3) Overexpression of p67 reduced phosphorylation of coexpressed eIF-2 alpha. (4) p67 synthesis was inhibited by cotransfection with an eIF-2 alpha mutant S51D, a mutant that mimics phosphorylated eIF-2 alpha, indicating that p67 cannot bypass translational inhibition mediated by phosphorylation of the eIF-2 alpha-subunit. These results show that the cellular protein p67 can reverse PKR-mediated translational inhibition in intact cells.     

Mutations in the alpha subunit of eukaryotic translation initiation factor 2 (eIF-2 alpha) that overcome the inhibitory effect of eIF-2 alpha phosphorylation on translation initiation

Phosphorylation of eIF-2 alpha in Saccharomyces cerevisiae by the protein kinase GCN2 leads to inhibition of general translation initiation and a specific increase in translation of GCN4 mRNA. We isolated mutations in the eIF-2 alpha structural gene that do not affect the growth rate of wild-type yeast but which suppress the toxic effects of eIF-2 alpha hyperphosphorylation catalyzed by mutationally activated forms of GCN2. These eIF-2 alpha mutations also impair translational derepression of GCN4 in strains expressing wild-type GCN2 protein. All four mutations alter single amino acids within 40 residues of the phosphorylation site in eIF-2 alpha; however, three alleles do not decrease the level of eIF-2 alpha phosphorylation. We propose that these mutations alter the interaction between eIF-2 and its recycling factor eukaryotic translation initiation factor 2B (eIF-2B) in a way that diminishes the inhibitory effect of phosphorylated eIF-2 on the essential function of eIF-2B in translation initiation. These mutations may identify a region in eIF-2 alpha that participates directly in a physical interaction with the GCN3 subunit of eIF-2B.     

Cultivation of cell-polymer cartilage implants in bioreactors

Primary T-cells are metabolically quiescent, with little DNA, RNA or protein synthesis. Upon mitogenic stimulation the rate of protein synthesis increases 10-fold. We have studied the role of eIF-2 and eIF-4 alpha (eIF-4E) expression in the mechanism of translational activation. During this period, the levels of eIF-2 alpha and eIF-4 alpha mRNA increase some 50-fold. Similar to the increase in ribosomes and mRNA, the number of eIF-2 alpha, eIF-2 beta, and eIF-4 alpha molecules per cell also increase 2-3-fold. This suggests that in addition to an increase in the pool size of translational components, an additional mechanism exists which results in an increased efficiency of factor utilization. We have looked at initiation factor phosphorylation. We find that eIF-2 alpha does not undergo significant changes in its phosphorylation state nor is there a change in the efficiency of eIF-2 utilization. However, there is a rapid increase in the phosphorylation state of eIF-4 alpha which correlates with the rapid increase in translational activity. It thus appears there are 2 distinct components responsible for the translational activation of quiescent T-cells during mitogenic stimulation. The first is the phosphorylation of eIF-4 alpha, with a concomitant increase in the efficiency of eIF-4 alpha utilization. The second is an increase in the pool sizes of eIF-2 and eIF-4 alpha.     

Removal of beta subunit of the eukaryotic polypeptide chain initiation factor 2 by limited proteolysis

It is generally considered that the eukaryotic polypeptide chain initiation factor 2 (eIF-2) from rabbit reticulocytes consists of three nonidentical subunits termed alpha, beta, and gamma, in order of increasing molecular weight. However, a recent report [Stringer, E. A., Chaudhuri, A., Valenzuela, D. & Maitra, U. (1980) Proc. Natl. Acad. Sci. USA 77, 3356-3359] suggested that this factor is made up of only two subunits. In this paper we show that limited proteolysis of rabbit reticulocyte eIF-2 leads to loss of the beta subunit. This modified eIF-2 has the same activity as the native factor in promoting ternary (eIF-2.GTP.Met-tRNAi) and 40S (eIF-2.GTP.Met-tRNAi.40S ribosome) initiation complex formation. Like native eIF-2, the protease-treated factor can restore translation in heme-deficient lysates. On the other hand, the treated factor is less stable than the native protein.     

The alpha-subunit of the mammalian guanine nucleotide-exchange factor eIF-2B is essential for catalytic activity in vitro

Eukaryotic initiation factor (eIF)-2B, the guanine nucleotide exchange factor for eIF-2, consists of five distinct subunits in both mammals and the yeast Saccharomyces cerevisiae. The exchange reaction mediated by eIF-2B can be regulated by phosphorylation of eIF-2 on its alpha-subunit. This represents a key control point in the initiation of translation. The functions of the individual subunits of the eIF-2B complex remain unclear. Mutational analysis in Saccharomyces cerevisiae suggested that the smallest subunit (the alpha) is dispensable for exchange, but required for the inhibition of eIF-2B by eIF-2(alphaP). Here we present evidence that, in mammalian cells, eIF-2Balpha is essential for the activity of the complex, since preparations of eIF-2B lacking this subunit are not active in nucleotide exchange in vitro, although the complex still contains the beta, gamma, delta and epsilon subunits.     

Use of monoclonal antibodies to study the structure and function of eukaryotic protein synthesis initiation factor eIF-2B

The eukaryotic protein synthesis initiation factor, eIF-2B, is a multimeric protein of five different subunits termed alpha, beta, gamma, delta and epsilon, which facilitates recycling of a further factor, eIF-2, and is an important control point in the initiation process. In order to investigate the structure and function of eIF-2B, monoclonal antibodies have been prepared to the beta, delta and epsilon subunits of the factor from rabbit reticulocytes. All three antibodies are active in Western blotting, ELISA and immunoprecipitation. The anti-epsilon antibody inhibits both the guanine nucleotide exchange activity of eIF-2B and protein synthesis in the rabbit reticulocyte lysate at the level of initiation. The other two antibodies do not inhibit either guanine nucleotide exchange or protein synthesis. The monoclonal antibodies and a polyclonal anti-(rabbit reticulocyte eIF-2B) serum were used to investigate the subunit size and the antigenic structure of eIF-2B from a variety of rabbit tissues and from a variety of mammalian species. eIF-2B from all rabbit tissues tested was indistinguishable from that prepared from rabbit reticulocytes. Quantitative studies showed substantial variation in the relative concentrations of eIF-2 and eIF-2B between different rabbit tissues. Marked variation in both the sizes of the subunits and their reaction with the antibodies was observed between eIF-2B from rabbit, rat, guinea pig and man.     

Expression of a phosphorylation-resistant eukaryotic initiation factor 2 alpha-subunit mitigates heat shock inhibition of protein synthesis

Protein synthesis is dramatically reduced upon exposure of cells to elevated temperature. Concordant with this inhibition, multiple phosphorylation and dephosphorylation reactions occur on specific eukaryotic initiation factors that are required for protein synthesis. Most notably, phosphorylation of the alpha-subunit of eukaryotic initiation factor-2 (eIF-2 alpha) on serine residue 51 occurs. To identify the importance of phosphorylation in control of protein synthesis, we have evaluated the effects of expression of a mutant eIF-2 alpha which is resistant to phosphorylation. Expression of a serine to alanine mutant at residue 51 of eIF-2 alpha partially protected cells from the inhibition of protein synthesis in response to heat treatment. The overexpressed serine to alanine 51 mutant subunit was incorporated into the eIF-2 heterotrimer and was resistant to phosphorylation. These results are consistent with the hypothesis that heat shock inhibition of translation is mediated in part through phosphorylation of eIF-2 alpha. Expression of the wild type or mutant eIF-2 alpha did not affect cell survival or induction of hsp70 mRNA upon heat shock, indicating that although eIF-2 alpha is a heat shock-induced protein, its increased synthesis during heat shock does not alter the heat-shock response.     

GTP hydrolysis controls stringent selection of the AUG start codon during translation initiation in Saccharomyces cerevisiae

We have isolated and characterized two suppressor genes, SUI4 and SUI5, that can initiate translation in the absence of an AUG start codon at the HIS4 locus in Saccharomyces cerevisiae. Both suppressor genes are dominant in diploid cells and lethal in haploid cells. The SUI4 suppressor gene is identical to the GCD11 gene, which encodes the gamma subunit of the eIF-2 complex and contains a mutation in the G2 motif, one of the four signature motifs that characterizes this subunit to be a G-protein. The SUI5 suppressor gene is identical to the TIF5 gene that encodes eIF-5, a translation initiation factor known to stimulate the hydrolysis of GTP bound to eIF-2 as part of the 43S preinitiation complex. Purified mutant eIF-5 is more active in stimulating GTP hydrolysis in vitro than wild-type eIF-5, suggesting that an alteration of the hydrolysis rate of GTP bound to the 43S preinitiation complex during ribosomal scanning allows translation initiation at a non-AUG codon. Purified mutant eIF-2gamma complex is defective in ternary complex formation and this defect correlates with a higher rate of dissociation from charged initiator-tRNA in the absence of GTP hydrolysis. Biochemical characterization of SUI3 suppressor alleles that encode mutant forms of the beta subunit of eIF-2 revealed that these mutant eIF-2 complexes have a higher intrinsic rate of GTP hydrolysis, which is eIF-5 independent. All of these biochemical defects result in initiation at a UUG codon at the his4 gene in yeast. These studies in light of other analyses indicate that GTP hydrolysis that leads to dissociation of eIF-2 x GDP from the initiator-tRNA in the 43S preinitiation complex serves as a checkpoint for a 3-bp codon/anticodon interaction between the AUG start codon and the initiator-tRNA during the ribosomal scanning process.     

Homozygous deletions of the CDKN2 gene and loss of heterozygosity of 9p in primary hepatocellular carcinoma

To examine the role of phosphorylation of the elongation factor eEF-1 in regulation of translation, 32P-labeled 3T3-L1 cells were deprived of serum, then incubated in the presence or absence of 10 nM insulin for 15 min. eEF-1 was purified by affinity chromatography on tRNA-Sepharose and shown to be phosphorylated on the alpha, beta and delta subunits. Phosphorylation of eEF-1alpha was stimulated sixfold in response to insulin, beta was stimulated fourfold and delta was threefold. The rate of elongation assayed with eEF-1 from insulin-stimulated cells was over twofold greater than with eEF-1 from serum-deprived cells. When eEF-1 from insulin-treated cells was subjected to two-dimensional tryptic phosphopeptide mapping, nine phosphopeptides were obtained with the alpha subunit, one with the beta subunit and three with the delta subunit. When compared with phosphopeptide maps of alpha, beta and delta subunits of eEF-1 phosphorylated in vitro by the insulin-stimulated multipotential protein kinase, the maps of the beta and delta subunits were identical. Five phosphopeptides obtained with the alpha subunit in vivo were identical to those obtained with S6 kinase in vitro; the remainder were unique. To examine whether protein kinase C had a role in phosphorylation of eEF-1 in response to insulin, protein kinase C was down-regulated by prolonged exposure of 3T3-L1 cells to 4beta-phorbol 12-myristate 13-acetate (PMA). Phosphorylation of the alpha, beta and delta subunits was stimulated 2.5-fold in response to insulin, with elongation activity stimulated to a similar extent, suggesting that protein kinase C had no effect on stimulation of elongation in response to insulin. Thus, stimulation of eEF-1 activity in response to insulin appears to be mediated primarily by multipotential S6 kinase. This data is consistent with previous studies on stimulation of initiation via phosphorylation of initiation factors by multipotential S6 kinase [Morley, S. J. & Traugh, J. A. (1993) Biochemie (Paris) 95, 985-989].     

A ribosomal protein is required for translational regulation of GCN4 mRNA. Evidence for involvement of the ribosome in eIF2 recycling

In amino acid-starved yeast cells, inhibition of the guanine nucleotide exchange factor eIF2B by phosphorylated translation initiation factor 2 results in increased translation of GCN4 mRNA. We isolated a suppressor of a mutant eIF2B. The suppressor prevents efficient GCN4 mRNA translation due to inactivation of the small ribosomal subunit protein Rps31 and results in low amounts of mutant 40 S ribosomal subunits. Deletion of one of two genes encoding ribosomal protein Rps17 also reduces the amounts of 40 S subunits but does not suppress eIF2B mutations or prevent efficient GCN4 translation. Our findings show that Rps31-deficient ribosomes are altered in a way that decreases the eIF2B requirement and that the small ribosomal subunit mediates the effects of low eIF2B activity on cell viability and translational regulation in response to eIF2 phosphorylation.     

Ligand interactions with eukaryotic translation initiation factor 2: role of the gamma-subunit

Eukaryotic translation initiation factor 2 (eIF-2) comprises three non-identical subunits alpha, beta and gamma. In vitro, eIF-2 binds the initiator methionyl-tRNA in a GTP-dependent fashion. Based on similarities between eukaryotic eIF-2gamma proteins and eubacterial EF-Tu proteins, we previously proposed a major role for the gamma-subunit in binding guanine nucleotide and tRNA. We have tested this hypothesis by examining the biochemical activities of yeast eIF-2 purified from wild-type strains and strains harboring mutations in the eIF-2gamma structural gene (GCD11) predicted to alter ligand binding by eIF-2. The alteration of tyrosine 142 in yeast eIF-2gamma, corresponding to histidine 66 in Escherichia coli EF-Tu, dramatically reduced the affinity of eIF-2 for Met-tRNAi(Met) without affecting the k(off) value for guanine nucleotides. In contrast, non-lethal substitutions at a conserved lysine residue (K250) in the putative guanine ring-binding loop increased the off-rate for GDP, thereby mimicking the function of the guanine nucleotide exchange factor eIF-2B, without altering the apparent dissociation constant for Met-tRNAi(Met). For eIF-2[gamma-K250R], the increased off-rate also seen for GTP was masked by the presence of Met-tRNAi(Met) in vitro. In vivo, increasing the dose of the yeast initiator tRNA gene suppressed the slow-growth phenotype and reduced GCN4 expression in gcd11-K250R and gcd11-Y142H strains. These studies indicate that the gamma-subunit of eIF-2 does indeed provide EF-Tu-like function to the eIF-2 complex, and further suggest that the level of Met-tRNAi(Met) is critical for maintaining wild-type rates of initiation in vivo.     

Identification and characterization of pancreatic eukaryotic initiation factor 2 alpha-subunit kinase, PEK, involved in translational control

In response to various environmental stresses, eukaryotic cells down-regulate protein synthesis by phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (eIF-2alpha). In mammals, the phosphorylation was shown to be carried out by eIF-2alpha kinases PKR and HRI. We report the identification and characterization of a cDNA from rat pancreatic islet cells that encodes a new related kinase, which we term pancreatic eIF-2alpha kinase, or PEK. In addition to a catalytic domain with sequence and structural features conserved among eIF-2alpha kinases, PEK contains a distinctive amino-terminal region 550 residues in length. Using recombinant PEK produced in Escherichia coli or Sf-9 insect cells, we demonstrate that PEK is autophosphorylated on both serine and threonine residues and that the recombinant enzyme can specifically phosphorylate eIF-2alpha on serine-51. Northern blot analyses indicate that PEK mRNA is expressed in all tissues examined, with highest levels in pancreas cells. Consistent with our mRNA assays, PEK activity was predominantly detected in pancreas and pancreatic islet cells. The regulatory role of PEK in protein synthesis was demonstrated both in vitro and in vivo. The addition of recombinant PEK to reticulocyte lysates caused a dose-dependent inhibition of translation. In the Saccharomyces model system, PEK functionally substituted for the endogenous yeast eIF-2alpha kinase, GCN2, by a process requiring the serine-51 phosphorylation site in eIF-2alpha. We also identified PEK homologs from both Caenorhabditis elegans and the puffer fish Fugu rubripes, suggesting that this eIF-2alpha kinase plays an important role in translational control from nematodes to mammals.     

The oxazolidinone linezolid inhibits initiation of protein synthesis in bacteria

The oxazolidinones represent a new class of antimicrobial agents which are active against multidrug-resistant staphylococci, streptococci, and enterococci. Previous studies have demonstrated that oxazolidinones inhibit bacterial translation in vitro at a step preceding elongation but after the charging of N-formylmethionine to the initiator tRNA molecule. The event that occurs between these two steps is termed initiation. Initiation of protein synthesis requires the simultaneous presence of N-formylmethionine-tRNA, the 30S ribosomal subunit, mRNA, GTP, and the initiation factors IF1, IF2, and IF3. An initiation complex assay measuring the binding of [3H]N-formylmethionyl-tRNA to ribosomes in response to mRNA binding was used in order to investigate the mechanism of oxazolidinone action. Linezolid inhibited initiation complex formation with either the 30S or the 70S ribosomal subunits from Escherichia coli. In addition, complex formation with Staphylococcus aureus 70S tight-couple ribosomes was inhibited by linezolid. Linezolid did not inhibit the independent binding of either mRNA or N-formylmethionyl-tRNA to E. coli 30S ribosomal subunits, nor did it prevent the formation of the IF2-N-formylmethionyl-tRNA binary complex. The results demonstrate that oxazolidinones inhibit the formation of the initiation complex in bacterial translation systems by preventing formation of the N-formylmethionyl-tRNA-ribosome-mRNA ternary complex.     

Double-stranded RNA-activated protein kinase (PKR) is negatively regulated by 60S ribosomal subunit protein L18

The double-stranded RNA (dsRNA)-activated protein kinase (PKR) provides a fundamental control step in the regulation of protein synthesis initiation through phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (eIF-2alpha), a process that prevents polypeptide chain initiation. In such a manner, activated PKR inhibits cell growth and induces apoptosis, whereas disruption of normal PKR signaling results in unregulated cell growth. Therefore, tight control of PKR activity is essential for regulated cell growth. PKR is activated by dsRNA binding to two conserved dsRNA binding domains within its amino terminus. We isolated a ribosomal protein L18 by interaction with PKR. L18 is a 22-kDa protein that is overexpressed in colorectal cancer tissue. L18 competed with dsRNA for binding to PKR, reversed dsRNA binding to PKR, and did not directly bind dsRNA. Mutation of K64E within the first dsRNA binding domain of PKR destroyed both dsRNA binding and L18 interaction, suggesting that the two interactive sites overlap. L18 inhibited both PKR autophosphorylation and PKR-mediated phosphorylation of eIF-2alpha in vitro. Overexpression of L18 by transient DNA transfection reduced eIF-2alpha phosphorylation and stimulated translation of a reporter gene in vivo. These results demonstrate that L18 is a novel regulator of PKR activity, and we propose that L18 prevents PKR activation by dsRNA while PKR is associated with the ribosome. Overexpression of L18 may promote protein synthesis and cell growth in certain cancerous tissue through inhibition of PKR activity.     

Phosphorylation of elongation factor 1 and ribosomal protein S6 by multipotential S6 kinase and insulin stimulation of translational elongation

Stimulation of protein synthesis in response to insulin is concomitant with increased phosphorylation of initiation factors 4B and 4G and ribosomal protein S6 (Morley, S. J., and Traugh, J. A. (1993) Biochimie 75, 985-989) and is due at least in part to multipotential S6 kinase. When elongation factor 1 (EF-1) from rabbit reticulocytes was examined as substrate for multipotential S6 kinase, up to 1 mol/mol of phosphate was incorporated into the alpha, beta, and delta subunits. Phosphorylation of EF-1 resulted in a 2-2. 6-fold stimulation of EF-1 activity, as measured by poly(U)-directed polyphenylalanine synthesis. The rate of elongation was also stimulated by approximately 2-fold with 80 S ribosomes phosphorylated on S6 by multipotential S6 kinase. When the rates of elongation in extracts from serum-fed 3T3-L1 cells and cells serum-deprived for 1.5 h were compared, a 40% decrease was observed upon serum deprivation. The addition of insulin to serum-deprived cells for 15 min stimulated elongation to a rate equivalent to that of serum-fed cells. Similar results were obtained with partially purified EF-1, with both EF-1 and ribosomes contributing to stimulation of elongation. These data are consistent with a ribosomal transit time of 3.2 min for serum-deprived cells and 1.6 min following the addition of insulin for 15 min. Taken together, the data suggest that insulin stimulation involves coordinate regulation of EF-1 and ribosomes through phosphorylation by multipotential S6 kinase.     

Phosphorylation of plant eukaryotic initiation factor-2 by the plant-encoded double-stranded RNA-dependent protein kinase, pPKR, and inhibition of protein synthesis in vitro

Regulation of protein synthesis by eukaryotic initiation factor-2alpha (eIF-2alpha) phosphorylation is a highly conserved phenomenon in eukaryotes that occurs in response to various stress conditions. Protein kinases capable of phosphorylating eIF-2alpha have been characterized from mammals and yeast. However, the phenomenon of eIF2-alpha-mediated regulation of protein synthesis and the presence of an eIF-2alpha kinase has not been demonstrated in higher plants. We show that plant eIF-2alpha (peIF-2alpha) and mammalian eIF-2alpha (meIF-2alpha) are phosphorylated similarly by both the double-stranded RNA-binding kinase, pPKR, present in plant ribosome salt wash fractions and the meIF-2alpha kinase, PKR. By several criteria, phosphorylation of peIF-2alpha is directly correlated with pPKR protein and autophosphorylation levels. Significantly, pPKR is capable of specifically phosphorylating Ser51 in a synthetic eIF-2alpha peptide, a key characteristic of the eIF-2alpha kinase family. Taken together, these data support the concept that pPKR is a member of the eIF-2alpha kinase family. In addition, the inhibition of brome mosaic virus RNA in vitro translation in wheat germ lysates by the addition of double-stranded RNA, phosphorylated peIF-2alpha, meIF-2alpha, or activated human PKR suggests that plant protein synthesis may be regulated via phosphorylation of eIF-2alpha.     

Requirement for homologous rabbit reticulocyte initiation factor 3 for initiation of alpha- and beta-globin mRNA translation in a crude protozoal cell-free system

Experimental evidence showing specificity of rabbit reticulocyte initiation factor 3 (EIF-3) for selective initiation of mRNA translation is presented. A new cell-free system was developed from Crithidia fasciculata. The crude postmitochondrial supernatant fluid was treated with puromycin and 0.5 M KCl to dissociate mRNA from polysomes and ribosomes into subunits. The drug and salt were removed by gel filtration on Sephadex G-25. Additions of amino acids and energy source initiate protein synthesis. All synthesis starts at the initiation site. This treatment brought about a shift in MgCl2 optimum from 6 to 3 mM. Exogenously supplied rabbit reticulocyte globin mRNA is faithfully translated in this system. However, crithidial EIF-3 has a low affinity for globin mRNA as evidenced by a 6-fold increase in the rate of globin synthesis after the addition of rabbit reticulocyte EIF-3 in the range at which globin synthesis is linear to the amount of globin mRNA added to the system. It is also shown that in a reconstituted system in which ribosomal subunits are depleted from initiation factors, EIF-3 from rabbit reticulocytes has a higher affinity for globin mRNA, as measured by the formation of polysomes during the linear time of amino acid incorporation. These results are taken to indicate that initiation factor EIF-3 action should be considered as an enzyme catalyzed reaction for which various mRNAs serve as different substrate analogs. Therefore, specificity is most likely to be expressed as an affinity of enzyme to substrate and would show as rate difference rather than an all-or-none phenomenon.