Characterization of the Two eIF4A-binding Sites on Human eIF4G-1*

Nadia L. KorneevaDagger , Barry J. LamphearDagger §, F. L. Colby HenniganDagger , William C. Merrick, and Robert E. RhoadsDagger ||

From the Dagger  Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center, Shreveport, Louisiana 71130-3932 and the  Department of Biochemistry, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106-4935

Received for publication, July 18, 2000, and in revised form, October 13, 2000



    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Eukaryotic translation initiation factor 4G-1 (eIF4G) plays a critical role in the recruitment of mRNA to the 43 S preinitiation complex. eIF4G has two binding sites for the RNA helicase eIF4A, one in the central domain and one in the COOH-terminal domain. Recombinant eIF4G fragments that contained each of these sites separately bound eIF4A with a 1:1 stoichiometry, but fragments containing both sites bound eIF4A with a 1:2 stoichiometry. eIF3 did not interfere with eIF4A binding to the central site. Interestingly, at the same concentration of free eIF4A, more eIF4A was bound to an eIF4G fragment containing both eIF4A sites than the sum of binding to fragments containing the single sites, indicating cooperative binding. Binding of eIF4A to an immobilized fragment of eIF4G containing the COOH-terminal site was competed by a soluble eIF4G fragment containing the central site, indicating that a single eIF4A molecule cannot bind simultaneously to both sites. The association rate constant, dissociation rate constant, and dissociation equilibrium constant for each site were determined by surface plasmon resonance and found to be, respectively, 1.2 × 105 M-1 s-1, 2.1 × 10-3 s-1, and 17 nM for the central site and 5.1 × 103 M-1 s-1, 1.7 × 10-3 s-1, and 330 nM for the COOH-terminal site.



    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The initiation of translation of most eukaryotic mRNAs involves the sequential recruitment of Met-tRNAi, mRNA, and the 60 S ribosomal subunit to the 40 S ribosomal subunit, catalyzed by the various groups of initiation factors (1). One of the most highly regulated steps is the recruitment of mRNA, which requires recognition of the 5'-terminal m7GTP-containing cap and 3'-terminal poly(A) tract, unwinding of 5'-terminal secondary structure, and binding to the 43 S initiation complex. These steps are mediated by members of the eIF41 group of initiation factors (eIF4A, eIF4B, eIF4E, and eIF4G) as well as poly(A)-binding protein.

eIF4A is the prototypical member of the DEXD/H-box protein family of nucleic acid helicases (2, 3). It functions as an ATP-dependent, bi-directional RNA helicase and RNA-dependent ATPase (4-7). The gamma -phosphate on the bound nucleotide has been shown to mediate changes in eIF4A conformation and RNA affinity. ATP binding and hydrolysis produce conformational changes in eIF4A that alter the RNA-protein interactions (8, 9, 3). A current model for protein synthesis initiation envisions eIF4A in the role of unwinding mRNA secondary structure in the 5'-untranslated region to allow the 40 S ribosomal subunit to bind the mRNA and/or scan it for the first AUG. The observation that dominant negative variants of eIF4A inhibit translation is consistent with such a role (10). Interestingly, the RNA helicase activity of eIF4A is ~20 times greater when bound to eIF4G than as a free protein (6, 11).

There are at least two genes for eIF4G in humans (12-14), wheat germ (15), and yeast (16). In mammals, these are termed eIF4G-1 and eIF4G-2.2 eIF4G serves to colocalize initiation factors involved in mRNA recruitment to the 43 S initiation complex. It directly binds RNA (17-19), poly(A)-binding protein (13), eIF4E (20, 21), the 40 S-binding protein complex eIF3 (20), eIF4A (20, 22), and the eIF4E kinase Mnk1 (23, 24). eIF4G in complex with these factors has the effect of bringing together the 3'- and 5'-termini of the mRNA (via poly(A)-binding protein and eIF4E), facilitates the binding of 40 S ribosomal subunit to mRNA (via eIF3), and promotes unwinding of 5'-untranslated region secondary structure (via eIF4A).

Initial studies indicated that the NH2-terminal one-third of mammalian eIF4G binds poly(A)-binding protein (13) and eIF4E (20, 21), the central one-third binds eIF3 (20), and the COOH-terminal one-third binds eIF4A (20) and Mnk1 (23, 24). These portions of eIF4G correspond with cleavage products of picornaviral proteases (cpN (aa 1-634), cpC3 (aa 635-1041), and cpC2 (aa 1042-1560), respectively; Ref. 20), suggesting that they represent individual structural and functional domains of the eIF4G molecule (see Fig. 1). The central domain has high amino acid sequence homology to all eIF4G proteins, whereas the COOH-terminal domain is poorly conserved (25). Additional studies revealed a second eIF4A-binding site in the central domain (22). The central domain of eIF4G is sufficient to catalyze cap-independent binding of ribosomes to RNA (17) and cap-independent but 5'-end-dependent translation in vitro (26) and in vivo (27). The function of the COOH-terminal domain is less clear. Variant forms of eIF4G containing both of the intact eIF4A-binding sites are more active in formation of the 48 S initiation complex and stimulation of cap-dependent translation than forms containing only one site (28). This has led to the proposal that interaction of eIF4A with the central domain is necessary for translation, whereas interaction of eIF4A with the COOH-terminal domain plays a modulatory role (28).

A complex of initiation factors termed eIF4F can be isolated by treatment of the 100,000 × g ribosomal pellet with 0.5 M KCl (29, 30). The eIF4F complex from mammalian cells contains eIF4A, eIF4E, and eIF4G (29, 30), but the complex from wheat germ (31, 32), yeast (33, 34), and Drosophila (35) contains only eIF4E and eIF4G. Even in mammalian eIF4F preparations, the amount of eIF4A varies, and passage of eIF4F over a phosphocellulose column completely removes eIF4A (11, 36). The stoichiometries of the mammalian eIF4F subunits have not been reported. The discovery of a second eIF4A-binding site in eIF4G (22) raises the question of whether two eIF4A molecules can bind simultaneously or whether a single eIF4A binds to both sites, as suggested recently (28). Similarly, the affinities of eIF4A to the two sites in mammalian eIF4G have not been reported. Since binding of eIF4A to eIF4G serves not only to localize eIF4A in the vicinity of the ribosome and mRNA but also enhances its intrinsic helicase activity, we undertook an analysis of the two eIF4A-binding sites in human eIF4G.


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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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Materials-- m7GTP-Sepharose 4B, heparin-Sepharose CL-6B, and a Mono Q HR 5/5 column were obtained from Amersham Pharmacia Biotech. Econo-Pac® 10 DG disposable chromatography columns and the Bio-Rad protein assay kit were obtained from Bio-Rad. S-protein-agarose, S-protein-bacterial alkaline phosphatase conjugate, and the plasmid pET-32a(+) were obtained from Novagen (Madison, WI). Nickel nitrilotriacetic acid-agarose was obtained from Qiagen (Chatsworth, CA). Bovine serum albumin was purchased from Pierce. Isopropyl-beta -D-thiogalactoside was obtained from Indofine Chemical Company (Belle Mead, NJ).

Amino Acid and Nucleotide Nomenclature-- Several cDNAs for eIF4G have been described that differ in the predicted length of the NH2-terminal polypeptide sequences (12, 13, 37). The aa and nt numbers used in the current work correspond to the larger form of eIF4G that begins with MNTPSQ (13). In this numbering system, the primary entero- and rhinoviral 2A protease cleavage site (38) is located between Arg-641 and Gly-642 (nt 1823-1828) (see Fig. 1). Fragments of eIF4G expressed from recombinant plasmids are named using inclusive aa numbers. Thus, S-eIF4G(877-1078) would contain the eIF4G sequence from aa 877 to 1078 fused at the NH2 terminus to thioredoxin, a His6 sequence, and the S-peptide of RNase A (which increases the molecular mass by ~20 kDa) (see Fig. 1). The plasmid expressing this protein would be called pTS4G(877-1078).

Construction of Plasmids-- pTS4G(613-1560) was constructed by inserting a restriction fragment from SmaI (nt 1749 in eIF4G cDNA) to XhoI (polylinker) derived from pSK-HFCl (39) between the EcoRV and XhoI sites of pET-32a(+) and then deleting the sequence from the BglII (polylinker) to BamHI (nt 1938 in eIF4G cDNA) sites. pTS4G(613-1078) was constructed by deleting a fragment between SpeI (nt 3334 in eIF4G cDNA) and SpeI (polylinker) sites from plasmid pSK(-)4GT7EV (40), yielding the plasmid pSK(-)4GT7SI. The sequence from BamHI (nt 1938 in eIF4G cDNA) to NotI (polylinker) were removed from pSK(-)4GT7SI and inserted into plasmid pET-32a(+) at the BglII and NotI sites. pTS4G(1078-1560) was constructed by deleting a fragment from SpeI (polylinker) to SpeI (nt 3334 in eIF4G cDNA) from pSK-HFCl (39), religating the larger restriction fragment, and inserting a restriction fragment from NotI (polylinker) to XhoI (polylinker) derived from it between the NotI and XhoI sites of pET-32a(+). pTS4G(877-1078) was constructed by amplifying a sequence from pTS4G(613-1078) by 20 cycles of polymerase chain reaction using the primers 5'-AGCTCCATGGTAATGCATGACTGTGTGGTC-3' and 5'-CCGCTGAGCAATAACTAG-3', the latter being the T7 terminator primer 69337-1 from Novagen. The products were digested with NcoI and XhoI and inserted between the NcoI and XhoI sites of the pET-32a(+) plasmid. pTS4G(975-1078) was constructed the same way, except the primer 5'-AGCTCCATGGATAGACATCGAGAGCAC-3' was used together with the T7 terminator primer. pTS4G(1317-1560) was constructed by inserting a restriction fragment from the NcoI (nt 3845 in eIF4G cDNA) to XhoI (polylinker) sites of pCITE4Gwt (40) into pET-32a(+) between the NcoI and XhoI sites. The eIF4G sequences in these plasmid were verified by DNA sequencing at the Iowa State University facility.

Purification of Recombinant Proteins-- The eIF4G fragments S-eIF4G(613-1560), S-eIF4G(613-1078), S-eIF4G(877-1078), S-eIF4G(975-1078), S-eIF4G(1078-1560), and S-eIF4G(1317-1560) were expressed in Escherichia coli strain BL21(DE3)pLysS (Novagen) and purified by nickel nitrilotriacetic acid-agarose chromatography. Cells were grown to A600 = 0.4 at 37 °C (typically 1-liter cultures except for S-eIF4G(613-1560) and S-eIF4G(613-1078), for which 4-l cultures were grown), and then expression was induced with isopropyl-beta -D-thiogalactoside to a final concentration of 0.5-1 mM. Five hours after induction at 30 °C, cells were harvested and frozen in liquid N2. Thawed cell pellets were resuspended in lysis buffer containing CompleteTM protease inhibitor tablets (Roche Molecular Biochemicals) and disrupted by sonication. Cleared extracts were incubated with nickel nitrilotriacetic acid-agarose (0.5 ml) with rotation for 2 h at 4 °C. After several washes with buffer A (50 mM Tris-HCl, 300 mM NaCl, 5 mM beta -mercaptoethanol, 10% (v/v) glycerol, and 20 mM imidazole), the recombinant proteins were eluted with buffer A containing 200 mM imidazole.

In the case of S-eIF4G(613-1078), it was necessary to perform an additional purification step on heparin-Sepharose CL-6B. The eluate from nickel nitrilotriacetic acid-agarose was diluted with an equal volume of buffer B0 (20 mM Tris-HCl, 2 mM EDTA, 5 mM beta -mercaptoethanol, 0.1% (v/v) Tween 20, and 5% (v/v) glycerol, pH 7.5) to reduce the salt concentration and then applied to a heparin-Sepharose CL-6B column equilibrated with the same buffer except containing 100 mM KCl (buffer B100). The protein was eluted with buffer B400.

eIF4G(642-1560) (used as a standard) and eIF4G(642-1078) were produced by incubation of S-eIF4G(613-1560) and S-eIF4G(613-1078), respectively, with recombinant Coxsackievirus 2A protease at 50 µg/ml for 1 h at 4 °C (41). In the case of eIF4G(642-1560), the resultant COOH-terminal portion was further purified from the S-peptide-tagged NH2-terminal portion by adsorption of the latter to S-protein-agarose.

Before performing binding experiments with S-protein-agarose, purified eIF4G fragments and eIF4A were passed over desalting Econo-Pac® 10 DG disposable chromatography columns to replace the buffer with buffer C150 (20 mM HEPES-KOH, 150 mM KCl, 2 mM beta -mercaptoethanol, 0.1% (v/v) Tween 20, and 2 mM EDTA, pH 7.5) plus 5% (v/v) glycerol. Before SPR analysis, purified eIF4G fragments and eIF4A were passed over the same columns except equilibrated with buffer D (20 mM HEPES-KOH, 150 mM KCl, 2 mM EDTA, 0.05% Tween 20, and 0.5 mM beta -mercaptoethanol, pH 7.5). After buffer exchange, the concentrations of recombinant proteins were determined with the Bio-Rad protein assay kit using bovine serum albumin as standard.

eIF3, eIF4A, and eIF4F Purification-- Purification and 14C-labeling of eIF4A by reductive methylation was performed as described previously (42). The particular batch of [14C]eIF4A used in these experiments was labeled to a specific activity of 187 cpm/pmol. eIF3, eIF4A, and eIF4F were purified from the ribosomal high salt wash of rabbit reticulocyte lysate by m7GTP-Sepharose and Mono Q chromatography (43). The eIF4A peak was rechromatographed on Mono Q with a shallower salt gradient. The eIF3 peak from the initial Mono Q chromatography was further purified by gel filtration on a SW300 column (Waters, Milford, MA) in buffer C150 plus 5% (v/v) glycerol.

Protein Binding Assays on S-protein-agarose-- Binding of S-eIF4G fragments with eIF3 and eIF4A was performed using S-protein-agarose. After 40 min of preincubation of S-eIF4G fragments with eIF3 or eIF4A on ice, proteins were mixed with at least a 10-fold molar excess of S-protein-agarose and incubated for 2 h in buffer C150 containing 1% milk proteins at 4 °C. The resin was washed 4 times with 200-µl aliquots of buffer C150. Proteins were eluted in SDS-electrophoresis buffer and analyzed by SDS-PAGE (44), with detection by Coomassie Blue staining, Western blotting, or autoradiography.

Antibodies-- Mouse monoclonal anti-eIF4A antibodies were a gift from Dr. Hans Trachsel, Bern, Switzerland. Rabbit anti-human eIF4G peptide 9 (aa 809-822) antibodies were produced as described previously (20).

Western Blotting-- For immunoblotting, proteins were transferred after SDS-PAGE to an Immobilon-P membrane (Millipore, Bedford, MA) using a Mini Trans-Blot cell (Bio-Rad). The membrane was incubated with a 1:1000 dilution of the primary antibodies in 5% milk proteins in buffer E (20 mM Tris-HCl, 150 mM NaCl, and 0.05% Tween 20, pH 7.5) for 1 h at room temperature, washed three times for 10 min with buffer E, and incubated with secondary anti-mouse or anti-rabbit antibodies conjugated with alkaline phosphatase (Vector Laboratories, Inc., Burlingame, CA) at a dilution of 1:2000 in 5% milk proteins in buffer E for 1 h at room temperature. Blots were developed with the nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl-phosphate color development substrate (Promega, Madison, WI).

Quantitation of Binding Data-- Quantitation of eIF4G fragments and eIF4A separated by SDS-PAGE was performed using a ScanMaker III laser densitometer (Microtek) and ImageQuant software, Version 3.3 (Molecular Dynamics). Experimental data were compared with standard curves consisting of purified S-eIF4G fragments and eIF4A for which the concentration has been determined with the Bio-Rad protein assay kit. Curve fitting was performed using SigmaPlot software, Version 4.01 (SPSS, Inc.). In cases of eIF4A binding with S-eIF4G(613-1078) or S-eIF4G(1078-1560), the data were fit with an equation describing the Langmuir isotherm,


<UP>BR</UP>=n[<UP>eIF4A</UP>]<SUB>f</SUB>/(K<SUB>d</SUB>+[<UP>eIF4A</UP>]<SUB>f</SUB>), (Eq. 1)
where BR is the binding ratio, i.e. the molar ratio of bound eIF4A to the eIF4G fragment, n is number of eIF4A-binding sites on the eIF4G fragment, [eIF4A]f is the concentration of eIF4A not bound to the resin, and Kd is the dissociation equilibrium constant for the eIF4A·eIF4G complex. [eIF4A]f was calculated as the difference between total eIF4A and bound eIF4A. A nonlinear least squares fit was performed in which n and Kd were allowed to vary.

An equation that takes into account two dissimilar binding sites (45) was used in titrations involving S-eIF4G(613-1560),
<UP>BR</UP>=n<SUB>1</SUB>[<UP>eIF4A</UP>]<SUB>f</SUB>/(K<SUB>d<UP>1</UP></SUB>+[<UP>eIF4A</UP>]<SUB>f</SUB>)+n<SUB>2</SUB>[<UP>eIF4A</UP>]<SUB>f</SUB>/(K<SUB>d<UP>2</UP></SUB>+[<UP>eIF4A</UP>]<SUB>f</SUB>), (Eq. 2)
where n1 and Kd1 represent the number of binding sites and dissociation equilibrium constant for the first binding site, respectively, and n2 and Kd2 represent the number of binding sites and dissociation equilibrium constant for the second.

SPR Analysis of S-eIF4G·eIF4A Interactions-- SPR was carried out using a BIAcore 2000 instrument (Biacore, Inc., Piscataway, NJ). eIF4A was immobilized on a research grade CM5 sensor chip using the amino coupling kit supplied by the manufacturer in 10 mM sodium acetate, pH 4.5. The surface density of immobilized eIF4A was 200-300 RU. One RU corresponds to an immobilized protein density of 1 pg/mm2 (46). The portion of the sensor chip in the first flow cell, used as a control, was subjected to activation and blocking in the same way as the eIF4A-containing cells but without added protein. The signals generated in the control flow cell were subtracted from the experimental signals to correct for refractive index changes and nonspecific binding.

All kinetic experiments were carried out in buffer D at 25 °C and a flow rate of 50 µl/min for S-eIF4G(613-1078) and S-eIF4G(1078-1560) and 20 µl/min for S-eIF4G(975-1078). At least five different concentrations of each S-eIF4G fragment were injected for each experiment. The first injection contained buffer without the S-eIF4G fragment. Between injections, the surface was regenerated with buffer F (20 mM HEPES-KOH, 500 mM KCl, 3 mM EDTA, 0.1% Tween 20, and 2 mM beta -mercaptoethanol, pH 7.5) at a flow rate of 70 µl/min and contact time of 2 min, followed by buffer D for 1 min.

Kinetic and equilibrium constants were calculated using the curve-fitting facility of the BiaEvaluation software, Version 3 (Biacore, Inc.). Binding data were globally fit to the 1:1 Langmuir binding model (A+B right-left-harpoons  AB). For this model, the function that describes the response (Rt) as a function of time (t) during the injection phase is,
R<SUB>t</SUB>=R<SUB><UP>eq</UP></SUB>[1−<UP>exp</UP>(<UP>−</UP>(k<SUB>a</SUB><UP>C</UP>+k<SUB>d</SUB>)(t−t<SUB>0</SUB>))] (Eq. 3)
where Req is the response at equilibrium, C is the molar concentration of S-eIF4G fragment, t0 is time at the start of injection, and ka and kd are association and dissociation rate constants, respectively. For the post-injection phase, the response is,
R<SUB>t</SUB>=R<SUB>0</SUB><UP>exp</UP>(<UP>−</UP>k<SUB>d</SUB>(t−t<SUB>0</SUB>)) (Eq. 4)
where R0 and t0 are the values of Rt and t, respectively, when sample is replaced with buffer, and kd is the dissociation rate constant. Values for the statistical closeness of fit, chi 2, were always below 1, indicating that the simple 1:1 model of interaction correctly described the experimental data.


    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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Definition of eIF4A-binding Sites on eIF4G-- Two eIF4A binding domains have been described for mammalian eIF4G, one in the central domain (22) and the other in the COOH-terminal domain (20) (Fig. 1). Evidence has recently been presented that the central eIF4A-binding site is located between aa 672 and 970 (28). To study these two binding sites separately, we prepared various recombinant portions of eIF4G fused to an NH2-terminal tag containing the S-peptide of RNase A (Fig. 1). [14C]eIF4A was incubated with each of these S-tagged eIF4G fragments, and the resultant complex was captured on S-protein-agarose. Material bound to the resin was eluted, subjected to SDS-PAGE, and analyzed for the eIF4G fragment by Coomassie Blue staining (Fig. 2A) and for eIF4A by autoradiography (Fig. 2B). S-eIF4G(613-1078) and S-eIF4G(1078-1560) were observed to bind eIF4A (lanes 1 and 2), but the other S-eIF4G fragments did not (lanes 3-5).



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Fig. 1.   Schematic representation of recombinant eIF4G fragments used in this study. The aa residues present in each fragment are indicated in parentheses. Binding sites for other proteins are shown by the shaded and cross-hatched boxes. The previous studies on which this model is based have suggested that the eIF4A- and eIF3-binding sites in the central domain are partially overlapping. The site of cleavage by entero- and rhinovirus 2A proteases is shown by the arrow. Molecular masses of eIF4G fragments plus the sequence tags are shown to the right of each protein. PABP, poly(A)-binding protein.



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Fig. 2.   Binding of eIF4A to eIF4G fragments. [14C]eIF4A (0.6 µM) was incubated with S-eIF4G(613-1078) (0.7 µM), S-eIF4G(1078-1560) (0.5 µM), S-eIF4G(1317-1560) (3.7 µM), S-eIF4G(877-1078) (2.6 µM), S-eIF4G(975-1078) (2 µM), and S-protein-agarose in a volume of 30 µl. Material bound specifically to the resin was eluted and subjected to SDS-PAGE on 8% gels. A, Coomassie Blue staining. B, autoradiography.

Since both eIF4A and eIF4G are capable of binding RNA, there was a possibility that the observed binding was due to an RNA linker. Therefore, the experiment was repeated after treatment of the proteins with micrococcal nuclease to remove any E. coli RNA that may have copurified with the recombinant proteins. The results were not altered from those seen in Fig. 2 (data not shown), indicating that the binding occurs in an RNA-independent manner.

eIF4A and eIF3 Do Not Compete for Binding to the Central Domain of eIF4G-- The central domain binds eIF4A as well as eIF3 (20, 22). To test whether this domain can bind eIF4A and eIF3 simultaneously, we performed a competition experiment. Reaction mixtures contained S-eIF4G(613-1078), a 5-fold molar ratio of [14C]eIF4A to S-eIF4G(613-1078), and a 2-, 5-, or 10-fold molar ratio of eIF3 to S-eIF4G(613-1078) (Fig. 3, lanes 2, 3, and 4, respectively). After incubation, the S-eIF4G(613-1078) and any bound proteins were captured with S-protein-agarose. The results indicated that eIF3 was retained on the S-protein-agarose (Fig. 3A, lanes 2-4). Furthermore, there was no decrease in eIF4A binding when increasing amounts of eIF3 were added, whether measured by Coomassie Blue staining (Fig. 3A, lanes 2-4) or autoradiography (Fig. 3B, lanes 2-4). There was no nonspecific binding of eIF4A or eIF3 to the resin in the absence of the eIF4G fragment (Fig. 3, lane 8).



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Fig. 3.   eIF3 and eIF4A do not compete for binding to eIF4G(613-1078). S-eIF4G(613-1078) was incubated at a concentration of 0.09 µM with a 5-fold molar ratio of [14C]eIF4A and a 2-, 5-, or 10-fold molar ratio of eIF3 (lanes 2, 3, and 4, respectively). The same concentration of S-eIF4G(613-1078) was incubated with a 5-fold molar ratio of eIF3 and a 2-, 5-, and 10-fold molar ratio of [14C]eIF4A (lanes 5, 6, and 7, respectively). Controls included S-eIF4G(613-1078) incubated in the absence of factors (lane 1) and [14C]eIF4A and eIF3 incubated in the absence of S-eIF4G(613-1078) (lane 8). All samples were treated with a quantity of S-protein-agarose (15 µl) such that S-Protein was in a 10-fold molar excess over S-peptide-tagged eIF4G. A, Coomassie Blue staining. B, autoradiography.

Another series of reaction mixtures contained S-eIF4G(613-1078), a 5-fold molar ratio of eIF3 to S-eIF4G(613-1078), and a 2-, 5-, or 10-fold molar ratio of [14C]eIF4A to S-eIF4G(613-1078). Proteins were again captured with S-protein-agarose (Fig. 3, lanes 5, 6, and 7, respectively). The results indicated that increasing amounts of eIF4A did not decrease eIF3 binding to S-eIF4G(613-1078) (Fig. 3A). The amount of bound [14C]eIF4A did not increase as the concentration of added [14C]eIF4A was increased because at all points, it was in molar excess to S-eIF4G(613-1078). These data show that there is no competition between eIF3 and eIF4A for binding to the S-eIF4G(613-1078) and, therefore, that the central domain of eIF4G can bind eIF4A and eIF3 simultaneously.

eIF4G(613-1078) and eIF4G(1078-1560) Each Bind eIF4A with a Stoichiometry of 1:1 but with Different Affinities-- To estimate the stoichiometry and equilibrium binding constants of eIF4A with S-tagged eIF4G fragments, we carried out titration experiments using S-protein-agarose. S-eIF4G(613-1078) was incubated at a concentration of 0.3 µM with either no eIF4A or with a 0.2-5-fold molar ratio of eIF4A. After adsorption to S-protein-agarose, the material bound to the resin was eluted and analyzed by SDS-PAGE, Coomassie Blue staining, and autoradiography (Fig. 4A). One reaction mixture containing eIF4A but no eIF4G fragment indicated that nonspecific binding of eIF4A was negligible (lane 12). The binding data were quantitated using standard curves, and a nonlinear least squares fit of the experimental data was performed using Equation 1 (Fig. 4B). The results indicated that the number of eIF4A-binding sites on S-eIF4G(613-1078), n, was 1.1 and that the equilibrium dissociation constant, Kd, was 96 nM (Fig. 4B). A linear transform of the data indicates the presence of a single binding site (Fig. 4B, inset).



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Fig. 4.   Saturation analysis of eIF4A binding to eIF4G fragments. A, S-eIF4G(613-1078) was incubated at a concentration of 0.3 µM with eIF4A at 0, 0.071, 0.1, 0.12, 0.14, 0.16, 0.18, 0.2, 0.35, 0.6, and 1.15 µM (lanes 1-11, respectively). The S-eIF4G(613-1078) and bound eIF4A were captured with a 10-fold molar excess of S-protein-agarose, subjected to SDS-PAGE on an 8% gel, and visualized with Coomassie Blue and autoradiography. B, the amounts of S-eIF4G(613-1078) and eIF4A in A were quantitated by autoradiography and by scanning stained bands and comparing the signals to standard curves of the two purified proteins electrophoresed on the same gel. BR is the molar ratio of eIF4A to S-eIF4G(613-1078), and [eIF4A]f is the total eIF4A concentration minus the complexed eIF4A. The curve is a least squares fitting of Equation 1 to the data, in which n and Kd are allowed to vary. The curve shown corresponds to n = 1.1 and Kd = 96 nM. The coefficient of determination (R2) was 0.96. Inset, replot of the data as BR/[eIF4A]f versus BR. C, same as A except that S-eIF4G(1078-1560) was used at 0.6 µM, and the eIF4A concentrations were 0.3, 0.65, 1, 2.65, and 5.3 µM for lanes 1-5, respectively. D, quantitation of the data in C by the same method as described in B. The curve shown corresponds to n = 1.1 and Kd = 660 nM. The R2 value was 0.94. Inset, the data of D are replotted as in B, inset. E, analysis of binding of S-eIF4G(613-1560) to varying concentrations of eIF4A. The protocol was the same as in A, except that S-eIF4G(613-1560) was used at 0.075 µM; the concentrations of eIF4A were 0, 0.14, 0.18, 0.27, 0.36, 0.75, and 1.23 µM in lanes 1-7, respectively; and detection of S-eIF4G(613-1560) and eIF4A were by Coomassie blue staining. F, standard curves of purified eIF4G(642-1560) and eIF4A were run on a different part of the same gel and used to quantitate the values in E. The data are plotted as in B, but the line corresponds to Equation 2 with n1 = 1.1, n2 = 1.1, Kd1 = 96 nM, and Kd2 = 660 nM. Inset, the data of F are replotted as in B, inset.

A similar experiment was performed with S-eIF4G(1078-1560). The S-eIF4G(1078-1560) was incubated at a concentration of 0.6 µM with a 0.5-10-fold molar ratio of eIF4A to S-eIF4G(1078-1560) (Fig. 4C). The data were quantitated as described above. The nonlinear least-squares fit yielded n = 1.1 and Kd = 660 nM, indicating that eIF4A binds S-eIF4G(1078-1560) with a stoichiometry of 1:1 (Fig. 4D). Scatchard analysis confirmed the existence of a single binding site on S-eIF4G(1078-1560) (Fig. 4D, inset). These and several other similar experiments indicated that, within experimental error, the stoichiometry of eIF4A binding to both the central and COOH-terminal sites was 1:1.

Stoichiometry of Binding of eIF4A with eIF4G(613-1560)-- The forgoing result indicated that one molecule of eIF4A could bind to either the central or COOH-terminal sites of eIF4G. The question then arose as to whether binding to these sites is mutually exclusive. We therefore titrated an eIF4G fragment containing both the central and COOH-terminal sites [S-eIF4G(613-1560)] using the same methodology. S-eIF4G(613-1560) was incubated at a concentration of 0.075 µM with eIF4A at a 1-16-fold molar ratio (Fig. 4E). Material bound to the resin was analyzed by SDS-PAGE.

Unique values of n and Kd for each site cannot be determined from empirical saturation data when there are two dissimilar binding sites on the same molecule (45). We therefore computed a theoretical curve from Equation 2 using the n and Kd values for S-eIF4G(613-1078) (1.1 and 96 nM, respectively) and S-eIF4G(1078-1560) (1.1 and 660 nM, respectively) that were determined experimentally in Figs. 4, B and D. The theoretical curve was similar to the experimental data, but several points fell above the line (see below). In addition, the maximum eIF4A binding to S-eIF4G(613-1560) approached a stoichiometry of 2:1 (Fig. 4F). Binding of eIF4A to the S-eIF4G(543-1560) fragment was also performed. In two separate experiments, the binding of eIF4A to that fragment also approached a 2:1 stoichiometry (data not shown).

As expected, plotting the theoretical curve calculated from Equation 2 in the manner used to produce a linear transform of the data of Figs. 4, B and D, resulted in a curved line (Fig. 4F, inset). The fact that several of the points fall above the theoretical curve means that the affinity of eIF4A for an eIF4G fragment containing both sites is higher than predicted by Equation. 2, which merely sums the individual contributions of the two separate sites. This suggests that there is cooperativity between sites, i.e. that the binding of eIF4A to one site increases the affinity to the other. Further support for cooperative binding is provided in the following experiment.

The Presence of Two eIF4A-binding Sites Enhances the Affinity of eIF4A for eIF4G-- Cooperativity between sites would predict that an eIF4G fragment containing both sites would bind more eIF4A than the sum of binding to fragments containing each separate site. To test this, we individually incubated S-eIF4G(613-1078) (central site), S-eIF4G(1078-1560) (COOH-terminal site), and S-eIF4G(613-1560) (both sites), each at 0.1 µM, with varying concentrations of eIF4A. The eIF4G fragments and bound eIF4A were then captured with S-protein-agarose and analyzed by Western blotting (Fig. 5). eIF4A incubated with a protein consisting of all elements at the NH2 terminus of the S-eIF4G fusion proteins (thioredoxin, His6-tag, and S-tag) but no eIF4G sequences was not retained on the resin (data not shown). At 0.39 µM eIF4A, the BR of eIF4A to S-eIF4G(1078-1560) was only 0.02, whereas the BR to S-eIF4G(613-1078) was 0.65, which is expected from the higher affinity of the latter. The predicted BR for S-eIF4G(613-1560), if there is no cooperativity between sites, is the sum of these, or 0.67. The observed BR, however, was 1.35, representing a 2-fold enhancement. At 0.65 µM eIF4A, the cooperative effect was not as high, which is to be expected since the sites were more nearly saturated. The sum of the BR values for the individual sites was 0.68, but the observed BR for S-eIF4G(613-1560) was 1.16, a 1.7-fold enhancement. Finally, at the highest eIF4A concentration, 1.3 µM, where both eIF4A-binding sites were nearly saturated, the enhancement was only 1.2-fold. Thus, binding of eIF4A to an eIF4G fragment that contains both eIF4A-binding sites is more than additive. These observations support the idea of positive cooperativity between the two sites.



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Fig. 5.   Comparative binding of eIF4A to eIF4G fragments containing one or two eIF4A-binding sites. eIF4A at the indicated concentrations was incubated with S-eIF4G(1078-1560), S-eIF4G(613-1078), or S-eIF4G(613-1560), each at 0.1 µM. The S-eIF4G fragments and bound eIF4A were captured on S-protein-agarose, subjected to SDS-PAGE on a 10% gel, and quantitated with both Coomassie Blue and Western blotting using standard curves for each protein run on the same gel. S-eIF4G(613-1560) predicted (open bars) is calculated assuming that there is no cooperativity between sites and represents the sum of the binding ratios for S-eIF4G(1078-1560) and S-eIF4G(613-1078) at the same eIF4A concentration.

Kinetic Measurements of eIF4A-eIF4G Interactions Using SPR-- The assessment of binding properties by retention of eIF4A·S-eIF4G complexes on S-protein-agarose represents a nonequilibrium situation, because the agarose beads must be washed several times with buffer not containing eIF4A to reduce nonspecific binding. It is suitable for determination of stoichiometries because a theoretical maximal binding is approached with increasing eIF4A concentration. However, the technique produces a systematic overestimate of Kd values (underestimate of binding affinities) because the average eIF4A concentration during binding and washing is less than the concentration during binding alone. We therefore turned to SPR, a technique that is not subject to this potential source of error, for the determination of binding constants.

eIF4A was immobilized on a sensor chip by the amino-coupling procedure, and eIF4G fragments were passed over it. Binding of S-eIF4G(613-1078) was measured over a range of concentrations from 3 to 100 nM (Fig. 6A). Binding of S-eIF4G(1078-1560) was measured over 10 to 1000 nM (Fig. 6B). As a control, S-eIF4G(975-1078), which carries the same tags as two other eIF4G fragments but does not contain an eIF4A-binding site (Fig. 2), was passed over the immobilized eIF4A at concentrations ranging from 3 to 300 nM (Fig. 6C). The three sensograms confirmed that S-eIF4G(613-1078) and S-eIF4G(1078-1560) each contain an eIF4A-binding site, whereas S-eIF4G(975-1078) does not. Thus, the NH2-terminal amino acid sequence (thioredoxin, His6, and S-peptide) does not interfere with the measurement.



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Fig. 6.   Kinetic analysis of eIF4A binding to S-eIF4G(613-1078) and S-eIF4G(1078-1560) by SPR. A, buffer D containing either 0, 3, 10, 25, 50, or 100 nM S-eIF4G(613-1078) was passed over eIF4A immobilized on a sensor chip (successive curves from lowest to highest). At 280 s, the mobile phase was changed back to buffer D alone. Thin traces correspond to a theoretical fitting of the data with a simple 1:1 kinetic binding model using the ka and kd values given in D. A "global fit" was used in which the single best values of ka and kd were obtained for all concentrations of analyte. The sensor chip contained about 300 RU of immobilized eIF4A. B, the same as A except that 0, 10, 50, 250, 450, 600, and 1000 nM solutions of S-eIF4G(1078-1560) were passed over the sensor chip containing 250 RU of eIF4A. C, S-eIF4G(975-1078), which does not contain an eIF4A-binding site (see Fig. 2), was passed over a chip containing 200 RU eIF4A at concentrations of 0, 3, 10, 25, 50, 100, 200, and 300 nM. The response curves closest to the base line correspond to the lowest concentration. D, association (ka) and dissociation (kd) rate constants calculated from the data in A and B using a global fit of the 1:1 binding model. The dissociation equilibrium constant Kd is calculated as kd/ka. The chi 2 value is a statistical measure of the closeness of fit. Residuals are the maximal deviation between actual and theoretical responses.

The best fit to the experimental data for binding of S-eIF4G(613-1078) and S-eIF4G(1078-1560) to eIF4A was provided with the simple 1:1 binding model (thin lines in Figs. 6, A and B; see "Experimental Procedures"). The low values of the average residuals (deviation of actual from theoretical) and chi 2 (Fig. 6D) indicate that the 1:1 binding model provides a good fit to the data. The rate constants obtained for association (ka) and dissociation (kd) are shown in Fig. 6D. An apparent dissociation constant, Kd, can be calculated from kd/ka (Fig. 6D). However, it should be noted that there may be hidden intermediates in the binding of eIF4A to eIF4G, and the rate constants obtained only reflect the slowest step in this process. The results show that S-eIF4G(613-1078) associates with eIF4A about 24-fold faster than does S-eIF4G(1078-1560). S-eIF4G(613-1078) also dissociates from eIF4A 1.2-fold faster than does S-eIF4G(1078-1560). As a result, S-eIF4G(613-1078) has an affinity for eIF4A that is 19-fold higher than S-eIF4G(1078-1560). Kd values obtained by SPR are considerably lower than those obtained by S-protein-agarose for the same protein-protein interactions, (17 versus 96 nM for S-eIF4G(613-1078) and 330 versus 660 nM for S-eIF4G(1078-1560)). This is consistent with the fact that the S-protein-agarose protocol represents a nonequilibrium condition and is expected to underestimate binding affinities.

eIF4G(613-1078) and eIF4G(1078-1560) Compete for Binding to eIF4A-- The presence of two eIF4A-binding sites on eIF4G evokes two possible models regarding the domain or surface of eIF4A that binds each site. In Model 1, suggested previously (28), a single eIF4A molecule can bind simultaneously to both sites in eIF4G. This would occur if two different surfaces of eIF4A were bound to the two sites. This model is not contradicted by the finding of a 2:1 stoichiometry with S-eIF4G(613-1560) (Fig. 4F), since at sufficiently high eIF4A concentrations, a second eIF4A molecule could displace the first, thus converting an eIF4A·eIF4G complex, with two contacts between eIF4G and eIF4A, to a (eIF4A)2·eIF4G complex, with a single contact between eIF4G and each of the eIF4A molecules. In Model 2, the same surface of eIF4A binds to either site on eIF4G. In this model, binding of eIF4A to one site on eIF4G prevents its simultaneous binding to the other site. An alternative version of Model 2 that would predict the same results is that the two surfaces on eIF4A overlap each other.

To distinguish between these two models, we performed a competition experiment. One eIF4G fragment containing the COOH-terminal eIF4A-binding site and an S-tag, S-eIF4G(1078-1560), was incubated with [14C]eIF4A in the presence of increasing amounts of an eIF4G fragment containing the central eIF4A-binding site but no S-tag, eIF4G(642-1078) (the latter was produced by cleavage of S-eIF4G(613-1078) with Coxsackievirus 2A protease. Then the S-tagged eIF4G fragment was captured with S-protein-agarose (Fig. 7).



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Fig. 7.   Competition between eIF4G(642-1078) and S-eIF4G(1078-1560) for binding to eIF4A. eIF4G(642-1078), which contains no S-tag, was produced by treatment of S-eIF4G(613-1078) with Coxsackievirus 2A protease. S-eIF4G(1078-1560) (0.085 µM) was incubated with a 2-fold molar ratio of [14C]eIF4A and either no eIF4G(642-1078) (lane 2) or a 2-, 5-, or 10-fold molar ratio of eIF4G(642-1078) (lanes 3, 4, and 5, respectively). Lane 6 is the same as lane 4 except that 0.05 µM eIF3 was also present. Lanes 7-9 are the same as lanes 3-5 except that no S-eIF4G(1078-1560) was present. Lane 10 represents binding of S-eIF4G(613-1078) (the same molar concentration as eIF4G(642-1078) in lane 3) with eIF4A. Lane 1 is a control with [14C]eIF4A but no S-eIF4G(1078-1560). In all cases, the S-eIF4G(1078-1560) and bound eIF4A were captured by incubation with a 10-fold molar excess of S-protein-agarose and subjected to SDS-PAGE on a 10% gel. A, Coomassie Blue staining. B, Western blotting using anti-eIF4G peptide 9 antibodies. C, autoradiogram of [14C]eIF4A. Numbers under the lanes represent the molar ratio of eIF4A to S-eIF4G(1078-1560), expressed as a percentage of lane 2.

Two outcomes are predicted if Model 1 is correct. First, the untagged eIF4G fragment would be retained on S-protein-agarose in a "sandwich" [S-protein-agarose·S-eIF4G(1078-1560)·eIF4A·eIF4G(642-1078)]. In Model 2, no untagged eIF4G fragment should be bound to the resin. The second prediction is that addition of the untagged eIF4G fragment should not decrease the amount of eIF4A bound to the S-protein-agarose if Model 1 is correct. In fact, based on the observation of positive cooperativity between the two eIF4A-binding sites (Figs. 4F and 5), one would expect enhanced binding of eIF4A to the S-protein-agarose. Model 2, on the other hand, predicts that the untagged eIF4G fragment in solution would compete with the S-tagged eIF4G fragment for binding with eIF4A, resulting in reduction of eIF4A bound to the resin.

Reaction mixtures containing S-eIF4G(1078-1560), a 2-fold molar ratio of [14C]eIF4A to S-eIF4G(1078-1560), and untagged eIF4G(642-1078) in a 0-, 2-, 5-, and 10-fold molar ratio to S-eIF4G(1078-1560) were incubated and subjected to S-protein-agarose (Fig. 7, lanes 2, 3, 4, and 5, respectively). As shown in Fig. 7A, the amount of S-eIF4G(1078-1560) retained on S-protein-agarose was essentially constant, ranging from 2.9 to 3.1 pmol. The amount of untagged eIF4G(642-1078) retained on the resin was much less, ranging from 0.1 to 0.38 pmol (Fig. 7B, lanes 2-5). Comparison of Fig. 7B, lane 3 (eIF4G fragment treated with 2A protease) with Fig. 7B, lane 10 (the same molar amount as on lane 3, but the eIF4G fragment was not treated with Coxsackievirus 2A protease) shows that only 1.6% of the input competitor (0.1 pmol versus 6.1 pmol) was retained on the resin. Also, eIF4G(642-1078), a protein of about 50 kDa, was undetectable by Coomassie Blue staining (Fig. 7A, lanes 2-5 and lanes 7-9). The eIF4A retained on the resin was initially 0.98 pmol but decreased 6-fold to 0.17 pmol with increasing competitor (Fig. 7C, lane 2-5).

As a control, one tube contained S-eIF4G(1078-1560), [14C]eIF4A, untagged eIF4G(642-1078), and eIF3 (Fig. 7, lane 6). If S-eIF4G(1078-1560) and eIF4G(642-1078) were to make a sandwich via eIF4A, it would be possible to detect eIF3 bound to the S-protein-agarose, since eIF3 binds to eIF4G(642-1078) (20). However, there was no detectable eIF3 on the gel (Fig. 7A, lane 6). Another set of controls contained untagged eIF4G(642-1078) and [14C]eIF4A but no S-eIF4G(1078-1560) (lanes 7-9). The amount of eIF4G(642-1078) bound to the resin was low (0.03-0.46 pmol; Fig. 7B). Most significantly, there was no difference in the amount of eIF4G(642-1078) bound to the resin in the presence or absence of S-eIF4G(1078-1560) (Fig. 7B, lanes 3-5 versus 7-9), indicating that the binding of eIF4G(642-1078) to the resin was nonspecific. All these finding are consistent with Model 2 but not Model 1, indicating that the two eIF4A-binding sites on eIF4G compete with each other for binding to eIF4A and fail to form a sandwich with eIF4A.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In our earlier study on the domain structure of eIF4G, we presented evidence that eIF3 was bound to the central domain, whereas eIF4A was bound to the COOH-terminal domain (22). A subsequent study showed that eIF4A was also bound to the central domain (28). This raised the question of the relationship between the eIF3- and eIF4A-binding sites in the central domain. A previous study proposed that these two sites overlapped (28). This suggestion was based on the observation that mutations affecting eIF4A binding to the central region of eIF4G also caused loss of eIF3 binding (22, 28). Here we show that eIF3 and eIF4A do not compete for binding in the central domain, which suggests that the eIF4A- and eIF3-binding sites are distinct.

The stoichiometries of mammalian eIF4F subunits have not been previously determined. Furthermore, the relative amount of eIF4A in any given preparation of rabbit eIF4F varies.3 eIF4A is lost from the mammalian eIF4F complex by phosphocellulose chromatography (36, 11), Mono Q chromatography (43), and ultracentrifugation of eIF4F on sucrose gradients (20). For these reasons, merely measuring the relative amount of eIF4F subunits in purified preparations would not be expected to yield meaningful results. The approach we have taken here is to measure the direct interaction of purified eIF4A and recombinant eIF4G fragments. Titration of different S-tagged eIF4G fragments with eIF4A demonstrated that eIF4G fragments containing each of the individual eIF4A-binding sites bind eIF4A with a 1:1 stoichiometry, but fragments containing both sites bind eIF4A with a 1:2 stoichiometry.

We showed that the affinities of the two eIF4A-binding sites are different using two different methodologies, static binding on S-protein-agarose and kinetic binding using SPR. Both methods indicated that the central site has a higher affinity for eIF4A than the COOH-terminal binding site. The two methods did not agree quantitatively, however, with apparent Kd values by SPR being lower than those by S-protein-agarose. The SPR results are expected to be more accurate, since SPR measures binding and dissociation instantaneously, whereas the S-protein-agarose method measures only the protein bound after several washes to remove nonspecifically bound protein. Given the propensity of eIF4F to lose eIF4A during purification, it is not surprising that the apparent affinities as determined by SPR are higher. The fact that the best fit of the SPR data was provided by the 1:1 kinetic binding model provides further evidence for a 1:1 stoichiometry for each site.

The basis for the difference in affinities between the central and COOH-terminal eIF4A-binding sites is revealed by the kinetic results. Turnover of eIF4A occupancy of the central site is faster than that of the COOH-terminal site. eIF4A associates with eIF4G(613-1078) about 24-fold faster than with eIF4G(1078-1560) (ka = 1.21 × 105 versus 5.1 × 103 M-1 s-1), but it also dissociates from eIF4G(613-1078) 1.2-fold faster than from eIF4G(1078-1560) (kd = 2.1 × 10-3 versus 1.7 × 10-3 s-1). This more rapid turnover of eIF4A bound to the central site may have mechanistic consequences.

Our data suggest that the binding of two molecules of eIF4A to eIF4G occurs cooperatively. The amount of eIF4A bound to an eIF4G fragment containing both sites was greater than the sum of eIF4A bound to the individual sites, the effect being less pronounced as both sites became saturated (Fig. 5). Thus, the presence of one molecule of eIF4A on eIF4G may accelerate or stabilize binding of the other. This model is consistent with a recent observation that alteration of either one of the eIF4A-binding sites by site-directed mutagenesis results in a form of eIF4G that fails to bind to eIF4A, despite the presence of an intact second site (28). The proposed cooperativity between sites may also explain why eIF4F complexes purified from plant (31, 47, 48), yeast (33), and Drosophila (35) do not contain eIF4A, despite the fact that direct binding of eIF4A to eIF4G can be demonstrated in wheat germ (49, 50) and yeast (51, 52) and that the affinity of yeast eIF4G for eIF4A (Kd congruent  30 nM; Ref. 52) is comparable with that of the central site of human eIF4G (Kd = 17 nM; Fig. 6). These plant and yeast eIF4Gs have high homology to the central domain of human eIF4G but low homology to the COOH-terminal domain. Consequently, there may be no enhancement of eIF4A binding by the COOH-terminal domain in the nonmammalian eIF4Gs.

Recently the central domain of eIF4G was postulated to work as a "ribosome recruitment core," in concert with other factors, based on its ability to form 48 S pre-initiation complex and drive initiation of translation (17, 27, 28). The COOH-terminal domain was proposed to play a modulatory role in this process, since an eIF4G fragment containing both eIF4A-binding sites is more active than one in which the COOH-terminal site has been inactivated by site-directed mutagenesis (28). Similarly, UV-cross-linking of RNA to eIF4A is stimulated more by an eIF4G fragment containing both binding sites than by either one separately (17). Our observation that binding of eIF4A to eIF4G is cooperative could explain these results by ensuring a higher affinity interaction between eIF4G and eIF4A.

Two models of interaction of mammalian eIF4G with eIF4A have been proposed (28). One model would require that eIF4A have two different surfaces for binding to eIF4G, allowing eIF4A to be sandwiched between the central and COOH-terminal binding sites. It was suggested that a resultant change in eIF4G conformation makes it more active in translation. The other model proposes that the central domain of eIF4G is sterically hidden from free eIF4A by the COOH-terminal domain. In this model, eIF4A first binds to the COOH terminus and is later transferred to the central region (28). Both models propose a 1:1 stoichiometry of binding eIF4A and eIF4G. We show here that eIF4G fragments containing the individual eIF4A-binding sites compete with each other and fail to form a sandwich on eIF4A. This indicates that the determinants on eIF4A that are recognized by these two different binding sites are either the same or overlapping, which is incompatible with the first model. Titration experiments showed a 1:2 stoichiometry of binding of eIF4G to eIF4A, which is incompatible with the second model. Also, the kinetic experiments indicate that eIF4A binds faster to the central site than to the COOH-terminal site, contrary to the second model. Thus, our data allow us to propose a new model whereby eIF4G binds two molecules of eIF4A simultaneously. Furthermore, binding of these two molecules of eIF4A to eIF4G appears to occurs cooperatively. The implications of such a model on the concerted action of paired eIF4A molecules acting to accomplish the processive unwinding of mRNA remain to be explored.


    ACKNOWLEDGEMENTS

We thank Dr. Hans Trachsel for providing anti-eIF4A antibodies and Aili Cai, Minghua Chen, and Clint Waddell for valuable technical assistance.


    FOOTNOTES

* This work was supported by NIGMS, National Institutes of Health Grants GM20818 and GM26796.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Present address: ProdiGene, 101 Gateway Blvd., Suite 100, College Station, TX 77845.

|| To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center, 1501 Kings Hwy., Shreveport, LA 71130-3932. Tel.: 318-675-5161; Fax: 318-675-5180; E-mail: rrhoad@lsuhsc.edu.

Published, JBC Papers in Press, November 1, 2000, DOI 10.1074/jbc.M006345200

2 The nomenclature system recommended by an ad hoc committee appointed by the IUBMB is used here (53). However, since this work concerns only eIF4G-1, the term eIF4G will subsequently refer to eIF4G-1.

3 N. L. Korneeva and R. E. Rhoads, unpublished observations.


    ABBREVIATIONS

The abbreviations used are: eIF, eukaryotic initiation factor; aa, amino acid residues; BR, molar binding ratio of eIF4A to eIF4G fragments; PAGE, polyacrylamide gel electrophoresis; RU, response unit; SPR, surface plasmon resonance; nt, nucleotide(s).


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


1. Merrick, W. C., and Hershey, J. W. B. (1996) in Translational Control (Hershey, J. W. B. , Mathews, M. B. , and Sonenberg, N., eds) , pp. 31-69, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
2. de la Cruz, J., Kressler, D., and Linder, P. (1999) Trends Biochem. Sci. 24, 192-198[CrossRef][Medline] [Order article via Infotrieve]
3. Jankowsky, E., Gross, C. H., Shuman, S., and Pyle, A. M. (2000) Nature 403, 447-451[CrossRef][Medline] [Order article via Infotrieve]
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