Mammalian translation initiation factor eIF1 functions with eIF1A and eIF3 in the formation of a stable 40 S preinitiation complex.

We have examined the role of the mammalian initiation factor eIF1 in the formation of the 40 S preinitiation complex using in vitro binding of initiator Met-tRNA (as Met-tRNA(i).eIF2.GTP ternary complex) to 40 S ribosomal subunits in the absence of mRNA. We observed that, although both eIF1A and eIF3 are essential to generate a stable 40 S preinitiation complex, quantitative binding of the ternary complex to 40 S subunits also required eIF1. The 40 S preinitiation complex contained, in addition to eIF3, both eIF1 and eIF1A in a 1:1 stoichiometry with respect to the bound Met-tRNA(i). These three initiation factors also bind to free 40 S subunits, and the resulting complex can act as an acceptor of the ternary complex to form the 40 S preinitiation complex (40 S.eIF3.eIF1.eIF1A.Met-tRNA(i).eIF2.GTP). The stable association of eIF1 with 40 S subunits required the presence of eIF3. In contrast, the binding of eIF1A to free 40 S ribosomes as well as to the 40 S preinitiation complex was stabilized by the presence of both eIF1 and eIF3. These studies suggest that it is possible for eIF1 and eIF1A to bind the 40 S preinitiation complex prior to mRNA binding.

We have examined the role of the mammalian initiation factor eIF1 in the formation of the 40 S preinitiation complex using in vitro binding of initiator Met-tRNA (as Met-tRNA i ⅐eIF2⅐GTP ternary complex) to 40 S ribosomal subunits in the absence of mRNA. We observed that, although both eIF1A and eIF3 are essential to generate a stable 40 S preinitiation complex, quantitative binding of the ternary complex to 40 S subunits also required eIF1. The 40 S preinitiation complex contained, in addition to eIF3, both eIF1 and eIF1A in a 1:1 stoichiometry with respect to the bound Met-tRNA i . These three initiation factors also bind to free 40 S subunits, and the resulting complex can act as an acceptor of the ternary complex to form the 40 S preinitiation complex (40 S⅐eIF3⅐eIF1⅐eIF1A⅐Met-tRNA i ⅐eIF2⅐GTP). The stable association of eIF1 with 40 S subunits required the presence of eIF3. In contrast, the binding of eIF1A to free 40 S ribosomes as well as to the 40 S preinitiation complex was stabilized by the presence of both eIF1 and eIF3. These studies suggest that it is possible for eIF1 and eIF1A to bind the 40 S preinitiation complex prior to mRNA binding.
The initiation of translation in eukaryotic cells occurs by a sequence of partial reactions that require a number of specific proteins called eukaryotic (translation) initiation factors (eIFs). 1 According to the currently accepted view of translation initiation, primarily derived from in vitro studies with purified initiation factors, an obligatory intermediate step in the overall initiation reaction is the binding of the initiator Met-tRNA i as the Met-tRNA i ⅐eIF2⅐GTP ternary complex to a 40 S ribosomal subunit containing bound initiation factor eIF3. This interaction leads to the production of the 40 S preinitiation complex (40 S⅐eIF3⅐Met-tRNA i ⅐eIF2⅐GTP). The 40 S preinitiation complex then binds to the 5Ј-capped end of mRNA and scans the mRNA in a 5Ј33Ј direction until the 40 S complex encounters the initiating AUG codon to form the 40 S initiation complex (40 S⅐eIF3⅐mRNA⅐Met-tRNA i ⅐eIF2⅐GTP). This reaction requires the participation of three other initiation factors eIF4F, eIF4A, and eIF4B. Subsequently, the 60 S ribosomal subunit joins the 40 S complex in a reaction dependent on two other factors, eIF5 and eIF5B, to form a functional 80 S initiation complex (80 S⅐mRNA⅐Met-tRNA i ) (for review, see Refs. [1][2][3][4][5]. In addition to the initiation factors described above, the 17-kDa eIF1A and the 12-kDa eIF1 are also known to play essential roles in the overall initiation process (1)(2)(3)(4)(5). Earlier biochemical studies demonstrated that both eIF1 and eIF1A have a weak stimulatory effect on the binding of Met-tRNA i and mRNA to 40 S and 80 S initiation complexes in the presence of other factors (6 -11). The presence of eIF1A in the 40 S initiation complex was also shown in one of these earlier studies (9). In vivo studies in Saccharomyces cerevisiae demonstrated that the genes encoding these two small initiation factors are essential for initiation of protein synthesis and required for cell growth and viability (12)(13)(14). These observations are in accord with genetic studies in S. cerevisiae (15,16) that indicate that eIF1 (SUI 1) plays an essential role in the fidelity of start site selection. In a purified mammalian translation initiation system, Pestova et al. (17), using toe-printing analysis on natural ␤-globin mRNA, have reported that eIF1 in concert with eIF1A, promotes stable 40 S complex formation with ribosomes positioned at the correct AUG codon. The requirements of eIF1 and eIF1A in AUG selection were also observed by Algire et al. (18) in a reconstituted yeast translation initiation system containing a 43-nucleotide-long RNA with an AUG codon in the middle. However, the step at which eIF1 and eIF1A associate with the 40 S ribosomal subunits remains unclear. Are these initiation factors recruited following binding of the 40 S preinitiation complex to the 5Ј-capped end? Or, are both these factors recruited to the 40 S ribosomal subunit prior to the binding of the preinitiation complex to the 5Ј-end of the mRNA?
Recently we described (19,20) an in vitro translation initiation assay that specifically measures the transfer of Met-tRNA i (as Met-tRNA i ⅐eIF2⅐GTP ternary complex) to 40 S ribosomal subunits in the absence of mRNA or an AUG codon to form the 40 S preinitiation complex. Using this assay, we observed that both eIF1A and eIF3 were essential for the efficient transfer of Met-tRNA i ⅐eIF2⅐GTP ternary complex to 40 S ribosomal subunits to form a stable 40 S preinitiation complex (20). However, analysis of the 40 S preinitiation complex showed that, although eIF3 was bound to the complex, eIF1A was not. It was of interest, therefore, to examine whether eIF1 has any role in the formation of the preinitiation complex as well as in the stable association of eIF1A to this complex.
In the present work, we have used a modified initiation assay to show that, although eIF1, by itself, does not stimulate the formation of the 40 S preinitiation complex, at low concentrations of initiation factors, all three factors eIF1, eIF1A, and eIF3 are required for the quantitative binding of the Met-tRNA i ⅐eIF2⅐GTP ternary complex to 40 S ribosomal subunits to form the stable 40 S preinitiation complex. Furthermore, analysis of the in vitro assembled 40 S preinitiation complex shows that both eIF1 and eIF1A are present in a near 1:1 stoichiometry with respect to bound Met-tRNA i . We also demonstrate that these three initiation factors bind to 40 S ribosomal subunits in the absence of Met-tRNA i ⅐eIF2⅐GTP ternary complex and the resulting 40 S⅐eIF3⅐eIF1⅐eIF1A can then bind the Met-tRNA i ⅐eIF2⅐GTP ternary complex to form the 40 S preinitiation complex (40 S⅐eIF3⅐eIF1⅐eIF1A⅐Met-tRNA i ⅐eIF2⅐GTP). The implications of these results in relation to 40 S preinitiation complex formation are discussed.

EXPERIMENTAL PROCEDURES
tRNA, Ribosomal Subunits, Purified Proteins, and Antibodies-The preparation of 35 S-or 3 H-labeled rabbit liver initiator Met-tRNA i (10,000 and 66,000 cpm/pmol, respectively) and 40 S and 60 S ribosomal subunits from Artemia salina eggs were as described previously (21). Purified eIF2 and eIF3 from rabbit reticulocyte lysates and bacterially expressed recombinant human eIF1A protein (unlabeled or labeled with 35 S) were isolated as described elsewhere (19). Rabbit IgG antibodies specific for mammalian eIF1A were obtained as described previously (19), and total IgY antibodies specific for mammalian eIF3 subunits were isolated from egg yolks of laying hens immunized with purified rabbit reticulocyte eIF3 (22). Polyclonal antibodies against purified denatured eIF1 were prepared in rabbits following a procedure similar to that used previously for preparation of anti-eIF1A antibodies (19). Immunoblot analysis was as described previously (23). The mixture of protease inhibitors added to buffer solutions used during purification of recombinant proteins from bacterial cell extracts consisted of leupeptin (0.5 g/ml), pepstatin A (0.7 g/ml), aprotinin (2 g/ml), and freshly prepared phenylmethylsulfonyl fluoride (1 mM).

Expression of eIF1 in Escherichia coli and Purification of the Recombinant Protein-
The open reading frame of eIF1 cDNA (24) was synthesized by reverse transcription-polymerase chain reaction of HeLa poly(A) ϩ RNA using the Invitrogen kit and appropriate oligonucleotide primers corresponding to the N-terminal and C-terminal ends of the eIF1 open reading frame. These primers had NdeI/EcoR1 overhangs. The PCR product was sequenced to ensure error-free DNA synthesis and cloned into the NdeI/EcoRI sites of pET-5a plasmid (Novagen). This pET-5a-eIF1 expression vector was used to transform E. coli BL21(DE3) cells (Novagen). Transformants were then grown at 37°C in 1 liter of LB medium (25) containing 50 g/ml ampicillin to an A 600 of about 1.2, induced with 1 mM isopropyl-␤-D-thiogalactoside, and grown for an additional 2 h. The cells were harvested by centrifugation, washed with 0.9% NaCl, quick-frozen in a dry ice/ethanol bath, and stored at Ϫ70°C.
For purification of recombinant eIF1, frozen E. coli cells (5 g) were disrupted by sonication in a buffer containing 20 mM Tris-HCl, pH 7.5, 10 mM MgCl 2 , 50 mM KCl, and 1 mM EDTA, and the postribosomal supernatant was prepared as described previously for the isolation of recombinant eIF5 from overproducing E. coli cells (26). The postribosomal supernatant (170 mg of protein in 16 ml of total volume) was loaded onto a 60-ml bed volume of DEAE-cellulose column equilibrated in buffer A (20 mM Tris-HCl, pH 7.5, 1 mM dithiothreitol, 0.1 mM EDTA, and 10% glycerol) plus 50 mM KCl, and the column was washed with buffer A plus 50 mM KCl. eIF1 was virtually unretarded under these conditions and appeared in the initial wash along with the unretarded protein peak. Fractions containing the protein peak were pooled (26 mg of protein), adjusted to 0.1 M KCl and loaded onto a 9-ml bed volume of a phosphocellulose column equilibrated in buffer A plus 100 mM KCl. The column was washed with the same buffer until A 280 of the effluent was below 0.1. Bound eIF1 was then eluted from the column with a linear gradient (total volume, 72 ml) in buffer A from 100 mM KCl to 800 mM KCl. Fractions containing eIF1 (eluting at about 450 mM KCl) were pooled, dialyzed against buffer A plus 50 mM KCl to reduce the ionic strength to that of buffer A plus 100 mM KCl, and then applied to a 1-ml bed volume fast-protein liquid chromatography Mono Q column. The column was washed with 5 ml of buffer A plus 100 mM KCl, and bound proteins were eluted by a gradient elution (12.5 ml of total volume) from buffer A plus 100 mM KCl to buffer A plus 400 mM KCl. Fractions containing eIF1 (eluting at about 150 mM KCl) were pooled, dialyzed against buffer B (20 mM Tris-HCl, pH 7.5, 2.5 mM 2-mercaptoethanol, 100 mM KCl, 0.1 mM EDTA, and 50% glycerol), and stored at Ϫ20°C. The yield was about 1 mg of homogeneous protein. eIF1 was monitored at different purification steps by SDS-PAGE followed by Coomassie Blue staining as well as by immunoblot analysis using rabbit polyclonal anti-eIF1 antibodies (data not shown).
Preparation of 3 H-Labeled eIF1-E. coli BL21(DE3) harboring the pET-5a-eIF1 expression plasmid was grown at 37°C in 500 ml of M9 medium (25) containing 50 g/ml ampicillin to A 600 of about 1.0, induced with 1 mM isopropyl-␤-D-thiogalactoside, and then grown for an additional hour. The culture was then supplemented with 5 mCi of [ 3 H]leucine (Amersham Biosciences), and the cells were allowed to grow with vigorous aeration for an additional hour. The cells were then harvested by centrifugation, washed with ice-cold 0.9% NaCl, and then quick-frozen. Homogeneous [ 3 H]eIF1 (ϳ800 cpm/pmol of protein) was isolated from the frozen cells using the purification protocol described above for the isolation of non-radioactive recombinant eIF1.
Assay for Formation of the 40 S Preinitiation Complex-40 S preinitiation complex formation was measured by the binding of 35 S-or 3 H-labeled Met-tRNA i (as the Met-tRNA i ⅐eIF2⅐GTP ternary complex) to 40 S ribosomal subunits at 1 mM Mg 2ϩ in the absence of mRNA or AUG as follows. Reactions were carried out in two stages. In Stage 1, reaction mixtures (100 l each) containing 20 mM Tris-HCl, pH 7.5, 100 mM KCl, 5 mM 2-mercaptoethanol, 4 g of nuclease-free bovine serum albumin, 400 M GTP, 1.2 g of purified rabbit reticulocyte eIF2, and 8 pmol of either [ 35 S]Met-tRNA i (30,000 -60,000 cpm/pmol) or [ 3 H]Met-tRNA i (10,000 cpm/pmol) were incubated at 37°C for 4 min to promote the formation of the [ 35 S]Met-tRNA i ⅐eIF2⅐GTP or the [ 3 H]Met-tRNA i ⅐ eIF2⅐GTP ternary complex. Reaction mixtures were then chilled in an ice-water bath, and a 5-l aliquot of the reaction mixture was subjected to nitrocellulose membrane filtration to determine the amount of the ternary complex formed (27). In Stage 2, another set of reaction mixtures (50 l each) containing 20 mM Tris-HCl, pH 7.5, 100 mM KCl, 1 mM MgCl 2 , 2.5 mM 2-mercaptoethanol (Buffer B), 0.6 A 260 units of 40 S ribosomal subunits and, where indicated, 1.5-15 g of eIF3, 50 ng to 1 g of eIF1A, and 50 -500 ng of eIF1 were incubated at 37°C for 4 min and then supplemented with 95 l of the Stage 1 reaction mixture containing about 4 pmol of 35 S-or 3 H-labeled Met-tRNA i ⅐eIF2⅐GTP ternary complex. The Mg 2ϩ concentration of each reaction mixture (now 175 l each) was adjusted to 1 mM, and the reaction mixtures were incubated at 37°C for 4 min, chilled in an ice-water bath, and then layered onto a 5-ml 7.5-30% (w/v) sucrose density gradient containing buffer B and centrifuged at 48,000 rpm for 105 min in a SW 50.1 rotor. Fractions (200 -300 l) were collected from the bottom of each gradient, and the radioactivity in each fraction was determined in a liquid scintillation spectrometer. The efficiency of the 40 S preinitiation complex formation was calculated relative to 35  S]Met-tRNA i was used for ternary complex formation in Stage 1 and unlabeled eIF3 and eIF1A were used in Stage 2. Following incubation to form the 40 S preinitiation complex, the reactions were analyzed either by sucrose gradient centrifugation as described above or by Sephadex G-75 gel filtration. For analysis by sucrose gradient centrifugation, 0.2-to 0.3-ml fractions were collected from the bottom of each tube following centrifugation, and the radioactivity in each fraction was measured by liquid scintillation counting. The presence of eIF3 in each fraction was determined by Western blotting.
For analysis by Sephadex G-75 gel filtrations, chilled reaction mixtures were each applied to a 12-ml bed volume column of Sephadex G-75 previously equilibrated in 20 mM Tris-HCl, pH 7.5, 1 mM MgCl 2 , 100 mM KCl, 1 mM dithiothreitol, and 5% glycerol. The column was then developed with the same equilibrating buffer at 4°C. Fractions of 250 l were collected and assayed for 3 H and/or 35 S radioactivity by counting each fraction in Aquasol in a liquid scintillation spectrometer. The elution positions of the 40 S preinitiation complex, free eIF1A, eIF1, or eIF3 proteins, and free Met-tRNA i were determined separately in the same column.

RESULTS
Role of eIF1 in the Formation of the 40 S Preinitiation Complex-We have previously used an in vitro assay that measures the binding of Met-tRNA i (as the Met-tRNA i ⅐eIF2⅐GTP ternary complex) to 40 S ribosomal subunits in the absence of mRNA or AUG codon to form the 40 S preinitiation complex (19,20). Although these initiation assays were carried out in reactions containing 1 mM Mg 2ϩ , they were subsequently analyzed for the binding of Met-tRNA i to 40 S ribosomes employing sucrose gradients in buffers containing 5 mM Mg 2ϩ . Elevated levels of Mg 2ϩ are known to stabilize ribosomal binding of Met-tRNA i and have been used by all investigators to analyze 40 S and 80 S initiation complexes in vitro by sucrose gradient centrifugation (7-11, 19, 20). Using such an assay, we showed that two initiation factors, eIF1A and eIF3, are required for efficient formation of a stable 40 S preinitiation complex. Although eIF1A promotes the transfer of Met-tRNA i ⅐eIF2⅐GTP ternary complex to 40 S ribosomal subunits to form the 40 S preinitiation complex, the presence of eIF3 bound to the 40 S ribosomal subunits is required to stabilize the resulting complex under a variety of experimental conditions (20).
To determine if eIF1 has a role in the formation and/or stability of the 40 S preinitiation complex, we used a similar assay system (19,20) except that both the initiation reactions and the subsequent sucrose gradient centrifugation analysis were carried out in buffers containing 1 mM Mg 2ϩ . We reasoned that the presence of 5 mM Mg 2ϩ in sucrose gradient buffers might artificially stabilize the bound Met-tRNA i and thus prevent the identification of additional factors involved in the 40 S preinitiation complex formation. For this reason a preformed [ 35 S]Met-tRNA i ⅐eIF2⅐GTP ternary complex (3.5 pmol) was incubated, at physiological 1 mM Mg 2ϩ with 40 S ribosomal subunits and saturating levels of each of the three initiation factors, eIF1A, or eIF1, or eIF3, either alone or in combination. The products were analyzed by sucrose gradient centrifugation in buffers containing 1 mM Mg 2ϩ (Fig. 1). When tested alone, eIF1A was the most effective of the three initiation factors in supporting binding of Met-tRNA i to 40 S ribosomal subunits. Although eIF1A, by itself, promoted the transfer of about 1.6 pmol of Met-tRNA i , eIF3 or eIF1, when used alone, supported binding of only 0.6 and 0.35 pmol of Met-tRNA i , respectively, to 40 S ribosomes. When both eIF1A and eIF3 were present in the After adjusting the Mg 2ϩ concentration to 1 mM, they were incubated a second time at 37°C for 4 min, chilled, and then analyzed by sucrose gradient centrifugation in buffers containing 1 mM Mg 2ϩ ("Experimental Procedures"). Initiation factor additions were as follows; छ, no addition; ࡗ, eIF1; Ⅺ, eIF3; ‚, eIF1A; f, eIF1 plus eIF3; OE, eIF1A plus eIF3; E, eIF1A plus eIF1; q, eIF1A plus eIF1 plus eIF3. The ascending arrows indicate 35  preinitiation reaction, the binding of Met-tRNA i to 40 S ribosomal subunits was increased to about 2.4 pmol. Surprisingly, although eIF1, by itself, did not promote significant 40 S preinitiation complex formation, its presence in the eIF1A-mediated reaction caused a marked stimulation of preinitiation complex production. About 2.9 pmol of the Met-tRNA i was bound to 40 S ribosomes under these conditions accounting for Ͼ80% of the input Met-tRNA i ⅐eIF2⅐GTP ternary complex (Fig. 1). In the presence of all three initiation factors, eIF1A, eIF1, and eIF3, Met-tRNA i was quantitatively transferred to the 40 S particle to form the 40 S preinitiation complex (Fig. 1). Omission of eIF1A from such a reaction markedly decreased the binding of Met-tRNA i to 40 S ribosomal subunits (Fig. 1). These results suggest that, although eIF1A is primarily responsible for transferring Met-tRNA i (as Met-tRNA i ⅐eIF2⅐GTP ternary complex) to 40 S ribosomes, the efficiency of this reaction is maximal in the presence of all three initiation factors. It should be noted that, in the experiments presented in Fig. 1 Under these conditions, nearly identical molar amounts of 3 H and 32 P were bound to 40 S ribosomes (data not shown).
The above results were obtained using saturating levels of each of the three initiation factors. However, in vivo, these factors are presumably present in much lower concentrations. The stimulatory effect of eIF1 on eIF1A-promoted transfer of Met-tRNA i to 40 S ribosomes became more pronounced when 40 S preinitiation complex formation was measured in the absence of eIF3 as a function of eIF1 and eIF1A concentrations. As shown in Fig. 2, lower concentrations of either eIF1A or eIF1, when used alone, supported the 40 S preinitiation complex production inefficiently. However, the presence of both eIF1A and eIF1 in preinitiation reactions caused a marked increase in the level of the 40 S preinitiation complex.
The reactions described above were performed with isolated 40 S ribosomal subunits. However, in vivo, both 40 S and 60 S ribosomal subunits are present in the same milieu. Thus, to investigate stable 40 S preinitiation complex formation under more physiological conditions, i.e. in the presence of both 40 S and 60 S ribosomal subunits, a preformed [ 35 S]Met-tRNA i ⅐eIF2⅐GTP ternary complex was incubated with 40 S ribosomal subunits and various combinations of eIF1A, eIF1, and eIF3 to form the 40 S preinitiation complex. The products were then treated with 60 S ribosomal subunits after raising the Mg 2ϩ concentration to 5 mM, conditions that favor spontaneous association of the ribosomal subunits. Analysis by sucrose gradient centrifugation revealed that the 40 S preinitiation complex formed with either eIF1A or eIF1 was disrupted nearly completely by 60 S ribosomal subunits, whereas the preinitiation complex formed with eIF3 alone was moderately stable (Fig. 3). Furthermore, although eIF1A and eIF1 together promoted significant 40 S preinitiation complex formation, nearly 60% of the resulting complex was not stable in the presence of 60 S ribosomal subunits (Fig. 3). In contrast and in keeping with previous results from this laboratory (20), when eIF1A and eIF3 were used to form the 40 S preinitiation complex, ϳ70% of the complex was stable in the presence of 60 S ribosomal subunits. However, when all three initiation factors were used to form the 40 S preinitiation complex, 60 S riboso- mal subunits failed to destabilize the complex formed and nearly 100% of the preinitiation complex was stable (Fig. 3). Each reaction product was then subjected to sucrose gradient centrifugation. As shown in Fig. 4, a fraction of labeled eIF1  (panel A) and eIF1A (panel B) co-sedimented with Met-tRNA i bound to the 40 S particle. Based on the level of radioactive eIF1 and eIF1A bound to the 40 S preinitiation complex, each of these factors was present in the 40 S preinitiation complex in a stoichiometry of ϳ1:1 with respect to the bound initiator Met-tRNA (Fig. 4). Western blot analysis using anti-eIF3 antibodies also detected eIF3 in the preinitiation complex in agreement with previous results (20) (Fig. 4C). These findings indicate that all three initiation factors, eIF3, eIF1A, and eIF1, can associate with the 40 S preinitiation complex.
The requirements for the binding of eIF1 and eIF1A to the 40 S preinitiation complex were also examined using a similar assay (Fig. 5). The binding of eIF1 was dependent on the simultaneous presence of eIF3. Although omission of eIF1A resulted in a moderate decrease in the binding of eIF1 relative to Met-tRNA i bound in the 40 S preinitiation complex, in the absence of eIF3 the amount of bound eIF1 was drastically reduced (Fig. 5). In contrast, eIF1A, in the presence of eIF1, can bind the 40 S preinitiation complex in the absence of eIF3, although the efficiency of binding was greatly reduced as compared with the binding in the presence of eIF3. Additionally, omission of eIF1 led to a near-complete abolition of eIF1A binding to the 40 S ribosomes. These findings agree with our previous results (19,20), showing that, when the 40 S preinitiation complex was formed in reactions containing eIF1A and  6. Binding of eIF1 and eIF1A to the 40 S preinitiation complex: analysis by Sephadex G-75 gel filtration. Two sets of reaction mixtures (each containing three reactions) were prepared for the formation of the 40 S preinitiation complex. The complete system in each set was prepared as described under the legend to Fig. 5, except that in one set [ 3 H]eIF1 (see Fig. 5A) was used, whereas the other used [ 35 S]eIF1A (see Fig. 5B). Various omissions were as indicated. Following 40 S preinitiation complex formation ("Experimental Procedures"), each reaction mixture was passed through a 12-ml bed volume column of Sephadex G-75 ("Experimental Procedures"). The eluted fractions were assayed for 3 H or 35 S radioactivity. The positions at which the 40 S preinitiation complex, free [ 3 H]eIF1, and free [ 35 S]eIF1A eluted from the column were determined separately in the same column and are shown. eIF3 but lacking eIF1, eIF1A did not associate with the 40 S preinitiation complex.
In the experiments described above, it was somewhat surprising that, although eIF1, in the absence of eIF3, stimulated the eIF1A-promoted transfer of Met-tRNA i to 40 S ribosomal subunits, eIF1 was not associated with the resulting 40 S complex. Likewise, in the absence of eIF1, eIF1A was also not bound to the 40 S preinitiation complex. The possibility exists that the association of eIF1 and eIF1A to the 40 S preinitiation complex in the absence of eIF3 or eIF1, respectively, was much weaker than when all three factors were present. Because the binding analysis (Fig. 5) was carried out by sucrose gradient centrifugation, conditions known to exert considerable hydrostatic pressure on the sedimenting particles, it was likely that in the absence of eIF3 and eIF1, respectively, both eIF1 and eIF1A dissociated from the 40 S complex during the sucrose gradient centrifugation. To examine this possibility, binding analyses were also performed using gel filtration. For this purpose, the 40 S preinitiation complex was formed by incubating (a) the [ 35 S]Met-tRNA i ⅐eIF2⅐GTP ternary complex with 40 S ribosomes, [ 3 H]eIF1, and unlabeled eIF1A and eIF3 and (b) the [ 3 H]Met-tRNA i ⅐eIF2⅐GTP ternary complex with 40 S ribosomes, [ 35 S]eIF1A, and unlabeled eIF1 and eIF3. Each reaction product was then subjected to Sephadex G-75 gel filtration (Fig. 6). In each case, as expected, labeled Met-tRNA i bound to the 40 S ribosomes eluted from the column in the excluded volume (data not shown). In agreement with the results obtained by sucrose gradient analysis (Fig. 5), both [ 3 H]eIF1 and [ 35 S]eIF1A were detected in the excluded fraction (Fig. 6, A and D) consistent with their association with the 40 S preinitiation complex. In the absence of 40 S ribosomal subunits, neither radiolabeled Met-tRNA i (not shown) nor [ 3 H]eIF1 or [ 35 S]eIF1A was detected in the excluded material (Fig. 6, B and E). However, when the preinitiation complex was formed in the absence of eIF3, [ 3 H]eIF1 was still detected in the excluded material, although the amount of bound [ 3 H]eIF1 was drastically reduced in this case (Fig. 6, compare C with A). Likewise, in reactions containing [ 35 S]eIF1A, omission of eIF1 did not abolish the binding of [ 35 S]eIF1A. Rather, the binding was somewhat reduced (Fig. 6, compare F with D). In sucrose gradient centrifugation analysis (see Fig. 5), neither eIF1 nor eIF1A was associated with the 40 S preinitiation complex under these conditions. Taken together, these results show that eIF1 and eIF1A were still associated with the 40 S preinitiation complex in the absence of either eIF3 or eIF1, respectively. However, their association was considerably weakened under these conditions, resulting in the dissociation of eIF1 and eIF1A from the 40 S preinitiation complex during sucrose gradient centrifugation (due to the hydrostatic pressure exerted on the sedimenting particles during the centrifugation).
Interaction of eIF1 and eIF1A with Free 40 S Ribosomal Subunits-Several laboratories have reported that mammalian eIF3 binds to 40 S ribosomal subunits in vitro in the absence of all other initiation components and the resulting 40 S⅐eIF3 complex is stable to sucrose gradient centrifugation (20,28,29). There is also evidence that, in vivo, the majority of native 40 S ribosomal subunits contain bound eIF3 (30,31). We examined whether both eIF1 and eIF1A can also associate with free 40 S ribosomal subunits in the absence of the Met-tRNA i ⅐eIF2⅐GTP ternary complex. Experiments were carried out in which free 40 S ribosomal subunits were incubated with either [ 3 H]eIF1 and unlabeled eIF1A and eIF3, or with [ 35 S]eIF1A and unlabeled eIF1 and eIF3, in the absence of Met-tRNA i ⅐eIF2⅐GTP ternary complex, and the products were analyzed by Sephadex G-75 gel filtration. Under these conditions, both [ 3 H]eIF1 and [ 35 S]eIF1A were detected in the excluded fractions (Fig. 7, A and F). In the absence of 40 S ribosomal subunits, neither 35 S nor 3 H radioactivity was eluted in the excluded fractions (Fig. 7, B and G). These results suggest that both eIF1 and eIF1A bind to 40 S particles in the absence of bound Met-tRNA i ⅐eIF2⅐GTP ternary complex. Analysis of the requirements for the binding of eIF1 to 40 S ribosomal subunits shows that, in the absence of eIF3, binding of [ 3 H]eIF1 was abolished almost completely (Fig. 7D). In contrast, in reactions containing eIF3 but no eIF1A, there was still significant but reduced binding of [ 3 H]eIF1 to 40 S ribosomes (Fig. 7C). No detectable binding of [ 3 H]eIF1 to 40 S ribosomes was observed in the absence of both eIF1A and eIF3 (Fig. 7E).
The requirements for eIF1A binding to 40 S ribosomal subunits were also examined. In this case, the binding of [ 35 S]eIF1A was only somewhat reduced in the absence of either eIF1 (Fig. 7H) or eIF3 (Fig. 7I). Even in the absence of both eIF1 and eIF3, low but detectable levels of eIF1A were still bound to the 40 S ribosomal subunits (Fig. 7J). Taken together, these results suggest that the binding of eIF1 to 40 S ribosomal subunits requires the presence of eIF3 and that, although eIF1A alone can bind to 40 S ribosomal subunits, this binding is stimulated markedly in the presence of both eIF1 and eIF3. It should be noted that when the binding analyses presented in Fig. 7 (A and F) were carried out by sucrose gradient centrifugation, no binding of either [ 3 H]eIF1 or [ 35 S]eIF1A was observed (data not shown). These results indicate that the association of eIF1 and eIF1A with free 40 S ribosomal subunits is relatively weak. This is also reflected by the trailing of the radioactivity of eIF1 and eIF1A in the elution profiles from gel filtrations presented in Fig. 7.
To investigate whether preformed 40 S⅐eIF3⅐eIF1⅐eIF1A complex can serve as an acceptor for the Met-tRNA i ⅐eIF2⅐GTP ternary complex, we isolated 40 S⅐eIF3⅐eIF1⅐eIF1A free of unreacted reaction components by Sephadex G-75 gel filtration.
Incubation of this isolated complex with Met-tRNA i ⅐eIF2⅐GTP resulted in the formation of the 40 S preinitiation complex (Fig.  8). Furthermore, all three initiation factors, eIF1, eIF1A, and eIF3, remained bound to the 40 S preinitiation complex (Fig. 8). DISCUSSION In the initiation of protein synthesis, the binding of the initiator Met-tRNA (as Met-tRNA i ⅐eIF2⅐GTP ternary complex) to 40 S ribosomal subunits is an obligatory intermediate step in the selection of a start codon in mRNA. This binding, which occurs in the absence of mRNA, leads to the formation of the 40 S preinitiation complex.
In studies presented here, we have investigated the initiation factor requirements for formation of a stable 40 S preinitiation complex using translation initiation assays that differed from those used previously (20) in that, following initiation reactions at 1 mM Mg 2ϩ , subsequent analyses of 40 S preinitiation complex formation were performed at 1 mM Mg 2ϩ rather than 5 mM Mg 2ϩ . We show that, although eIF1A and eIF3 together (in the absence of eIF1) promoted efficient formation of the stable 40 S preinitiation complex, the transfer of Met-tRNA i (as Met-tRNA i ⅐eIF2⅐GTP ternary complex) was not quantitative. Quantitative transfer of Met-tRNA i was only observed when eIF1 was also present. Under the modified conditions described here, all three factors were associated with the 40 S preinitiation complex. Furthermore, the 40 S preinitiation complex formed in the presence of all three factors was completely stable even in the presence of 60 S ribosomal subunits. It appears that, at 1 mM Mg 2ϩ , eIF1 plus eIF1A promotes the  transfer of Met-tRNA i ⅐eIF2⅐GTP ternary complex to the 40 S ribosomal subunit, and this effect of eIF1 becomes more evident when lower concentrations of the factors are used in the assays. At these lower concentrations, although eIF1A and eIF1 by themselves cannot promote transfer of Met-tRNA i to 40 S ribosomal subunits, the presence of both these factors leads to a marked stimulation in 40 S complex formation. However, in the absence of eIF3, eIF1 was not detected in the resulting 40 S preinitiation complex (analyzed by sucrose gradient centrifugation). These observations indicated that, although eIF1 can carry out its function in the absence of eIF3, the presence of eIF3 is required for the stable association of eIF1 with the 40 S preinitiation complex. These results are in agreement with the observation (14,32) that, in S. cerevisiae, eIF3 and eIF1 interact and are present in a multifactor complex (33). Similar interaction between mammalian eIF1 and eIF3 has also been observed in glutathione S-transferase pull-down assays (34). In contrast to eIF1, the stable eIF1A binding to the 40 S preinitiation complex, measured by sucrose gradient centrifugation, was not observed when the complex was formed in the presence of eIF1A and eIF3. Rather, binding of eIF1A to the 40 S preinitiation complex was dependent on the presence of eIF1. This observation explains our previous results (19,20) that, although eIF1A, by itself or in the presence of eIF3, can promote significant 40 S preinitiation complex formation, the resulting 40 S complex did not contain eIF1A, because these assays were carried out in the absence of eIF1.
The results presented in this report show that eIF3, eIF1, and eIF1A can also bind to free 40 S ribosomal subunits to form the 40 S⅐eIF3⅐eIF1⅐eIF1A complex (Fig. 7). Here again, the binding of eIF1 to free 40 S subunits appears to be dependent on the presence of eIF3. Likewise, although eIF1A, by itself, can bind weakly to 40 S ribosomal subunits, maximal binding of eIF1A requires the presence of both eIF1 and eIF3. Finally, it is important to note that the 40 S⅐eIF3⅐eIF1⅐eIF1A complex, itself, can act as an acceptor of the Met-tRNA i ⅐eIF2⅐GTP ternary complex to form a stable 40 S preinitiation complex (Fig.  8). These findings suggest the possibility that the 40 S⅐eIF3⅐eIF1⅐eIF1A complex may act as an intermediate in the binding of the Met-tRNA i ⅐eIF2⅐GTP ternary complex to the 40 S subunits to generate the 40 S preinitiation complex. Furthermore, it is possible that both eIF1 and eIF1A, like eIF3, can bind the 40 S ribosomal subunit prior to message binding.
An important outcome of the present studies is the role of eIF3 and eIF1A in ribosomal subunit anti-association. Mammalian eIF3 has been reported to bind free 40 S ribosomal subunits in the absence of initiator Met-tRNA and other initiation factors (28) and prevent the Mg 2ϩ -dependent association between 40 S and 60 S ribosomal subunits to form 80 S ribosomes (29,30,35). We have, however, observed (20) that, although eIF3 binds stably to free 40 S ribosomal subunits in the absence of all other initiation components, the addition of 60 S ribosomal subunits to the 40 S⅐eIF3 complex resulted in the release of eIF3 from the 40 S ribosomal subunits with the concomitant formation of 80 S ribosomes. This was true even when eIF1A was included in the reaction (19,20). These results suggested that eIF3 and eIF1A may not be directly involved in the generation of free ribosomal subunits. The results presented in this report, however, show that eIF3 does have antiassociation factor activity in the context of the 40 S preinitia-tion reactions. In the absence of eIF3, 60 S subunits can displace the 40 S subunits present in the 40 S preinitiation complex and presumably associate with these 40 S subunits to form 80 S ribosomes (Fig. 3). The presence of eIF3 (along with eIF1A and eIF1) bound to the 40 S preinitiation complex prevents the displacement of 40 S subunits from the preinitiation complex by the 60 S subunits (Fig. 3). Thus, under these conditions, 80 S ribosomes cannot be formed.