JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M007525200 on October 5, 2000

J. Biol. Chem., Vol. 275, Issue 52, 41369-41376, December 29, 2000
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
275/52/41369    most recent
M007525200v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Korneeva, N. L.
Right arrow Articles by Rhoads, R. E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Korneeva, N. L.
Right arrow Articles by Rhoads, R. E.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Mutually Cooperative Binding of Eukaryotic Translation Initiation Factor (eIF) 3 and eIF4A to Human eIF4G-1*

Nadia L. Korneeva, Barry J. LamphearDagger, F. L. Colby Hennigan, and Robert E. Rhoads§

From the Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center, Shreveport, Louisiana 71130-3932

Received for publication, August 18, 2000, and in revised form, October 2, 2000

    ABSTRACT
TOP
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. The central region of eIF4G binds the ATP-dependent RNA helicase eIF4A, the 40 S binding factor eIF3, and RNA. In the present work, we have further characterized the binding properties of the central region of human eIF4G. Both titration and competition experiments were consistent with a 1:1 stoichiometry for eIF3 binding. Surface plasmon resonance studies showed that three recombinant eIF4G fragments corresponding to amino acids 642-1560, 613-1078, and 975-1078 bound eIF3 with similar kinetics. A dissociation equilibrium constant of ~42 nM was derived from an association rate constant of 3.9 × 104 M-1 s-1 and dissociation rate constant of 1.5 × 10-3 s-1. Thus, the eIF3-binding region is included within amino acid residues 975-1078. This region does not overlap with the RNA-binding site, which suggests that eIF3 binds eIF4G directly and not through an RNA bridge, or the central eIF4A-binding site. Surprisingly, the binding of eIF3 and eIF4A to the central region was mutually cooperative; eIF3 binding to eIF4G increased 4-fold in the presence of eIF4A, and conversely, eIF4A binding to the central (but not COOH-terminal) region of eIF4G increased 2.4-fold in the presence of eIF3.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The initiation of translation in eukaryotes requires multiple initiation factors that stimulate the binding of mRNA and Met-tRNAi1 to the 40 S ribosomal subunit to form the 48 S preinitiation complex (1). The binding of Met-tRNAi occurs as a ternary complex with eIF2 and GTP. The binding of mRNA is stimulated by the eIF4 factors (eIF4A, eIF4B, eIF4E, and eIF4G). Joining of the 60 S subunit to form the 80 S initiation complex requires hydrolysis of the GTP bound to eIF2, dissociation of the ternary complex, and release of the eIF2·GDP binary complex. eIF5 and eIF5B promote these events by stimulating GTP hydrolysis within the ternary complex bound to the 40 S ribosomal subunit (2). eIF1 and eIF1A act synergistically to mediate assembly of initiation complexes at the initiation codon (3).

eIF3 is a multisubunit complex that has been implicated in several aspects of 48 S complex formation. It binds the 40 S ribosomal subunit, stabilizes binding of the eIF2·GTP·Met-tRNAi ternary complex to the 40 S subunit, stimulates binding of mRNA to the 40 S subunit, and promotes dissociation of 80 S ribosomes into 40 S and 60 S subunits (4-6). Mammalian eIF3 contains 10 non-identical polypeptides termed p170, p116, p110, p66, p48, p47, p44, p40, p36, and p35 (7, 8). Five of these polypeptides have identifiable homologs in Saccharomyces cerevisiae (8, 9). Characterization of rabbit and human eIF3 indicates that the complex has a molecular mass of ~600 kDa and that the subunits are present in one copy per particle (10). At least five subunits of mammalian eIF3, p170, p116 or p110,2 p66, p47, and p44, bind RNA (11-17). Of these, p66 and p44 have been shown to bind 18 S ribosomal RNA (13, 15, 18). Mammalian eIF3 also cross-links to 40 S ribosomal subunit proteins (19). Mammalian eIF3 binds eIF1 via p110 (20), eIF4B via p170 (21), and eIF5 (22). Yeast eIF3 binds both eIF1 and eIF5 via Nip1p (9) and eIF4B via p33 (23). Mammalian eIF3 also interacts with the eIF4F complex (which consists of eIF4E, eIF4A, and eIF4G) via eIF4G (24). A model whereby eIF3 serves as a bridge between the 40 S ribosomal subunit and the eIF4F·mRNA complex has been postulated (24).

eIF4G is the central linking protein for all initiation factors known to be involved in mRNA recruitment to the ribosome (25). eIF4G binds the cap-binding protein eIF4E (24, 26, 27), PABP (28-30), eIF4A (24, 31-33), eIF3 (24), the eIF4E-kinase Mnk1 (34, 35), and both mRNA (36) and the EMCV IRES (37, 38). Binding of eIF4G to these proteins brings together the 5' and 3' termini of mRNA (via eIF4E and PABP), RNA-helicase activity (via eIF4A), and the 40 S ribosomal subunit (via eIF3) (24). Thus, these polypeptides collectively recognize the characteristic structures of mRNA, unwind mRNA secondary structure, and facilitate binding of the 40 S ribosomal subunit.

Mammalian eIF4G can be divided into three domains, roughly corresponding to cleavage site by picornaviral 2A proteases (39, 24). The NH2-terminal one-third contains the eIF4E- and PABP-binding sites. The central domain contains the binding sites for eIF3, RNA, and one of the two sites for eIF4A. The COOH-terminal domain contains a second eIF4A-binding site and also a site for Mnk1. The central region of eIF4G serves as autonomous "ribosome recruitment core" in vivo (40) and in vitro (38), whereas the COOH-terminal domain has been proposed to serve as regulatory domain (41, 42).

Despite the exceptional progress that has been made in identifying ligands for this pivotal initiation factor, little is known about whether binding of one ligand to eIF4G influences the binding of others. Such knowledge may provide insight into the ordered series of events that results in proper placement of Met-tRNAi at the initiation codon. As a first step toward understanding the relationships between the various eIF4G ligands, we have more precisely defined the binding sites, developed methods to measure rates and stoichiometries of binding, and studied the mutual influence of eIF4A and eIF3 on their binding to eIF4G. Our data provide evidence that binding of eIF3 and eIF4A to the central domain of eIF4G occurs in a cooperative manner.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- m7GTP-Sepharose, heparin-Sepharose CL-6B, and a Mono Q column were obtained from Amersham Pharmacia Biotech (Piscataway, NJ). Econo-Pac 10 DG disposable chromatography columns and a protein assay kit were obtained from Bio-Rad Laboratories (Hercules, CA). S-protein-agarose, S-protein-bacterial alkaline phosphatase conjugate, and the plasmid pET32A(+) were obtained from Novagen (Madison, WI). Nickel-nitrilotriacetic acid-agarose was obtained from Qiagen (Chatsworth, CA). Protease inhibitor tablets (Complete) were obtained from Roche Molecular Biochemicals. Bovine serum albumin was purchased from Pierce (Rockford, IL). Isopropyl-beta -D-thiogalactoside was obtained from Indofine Chemical Co. (Belle Mead, NJ).

Construction of Plasmids-- Preparation of the plasmids pTS4G-(613-1560), pTS4G-(613-1078), pTS4G-(1078-1560), pTS4G-(877-1078), and pTS4G-(975-1078) is described elsewhere (42).

eIF3, eIF4A, and eIF4F Preparation-- Purification and 14C labeling of eIF4A by reductive methylation was performed as described previously (43). eIF3, eIF4A, and eIF4F were purified from the ribosomal high salt wash of rabbit reticulocyte lysate by m7GTP-Sepharose and Mono Q chromatography (44). 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 an SW300 column (Waters, Milford, MA) in buffer A (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.

Purification of Recombinant Proteins-- Some recombinant human eIF4G fragments contained NH2-terminal tags consisting of thioredoxin, His6, and the S-peptide of RNase A and COOH-terminal His6 tags, contributing an additional ~20 kDa to the proteins. The names of the proteins and their inclusive amino acid (aa) numbers of eIF4G (42) are as follows in parentheses: S-eIF4G-(613-1560), S-eIF4G-(613-1078), S-eIF4G-(877-1078), S-eIF4G-(975-1078), and S-eIF4G-(1078-1560). These were expressed in Escherichia coli strain BL21(DE3)pLysS (Novagen) and purified as described elsewhere (42) by nickel-nitrilotriacetic acid-agarose chromatography and, in the case of S-eIF4G-(613-1078), heparin-Sepharose CL-6B. eIF4G-(642-1560) was produced by proteolytic cleavage of S-eIF4G-(613-1560) using recombinant Coxsackievirus 2A protease (45) at 50 µg/ml for 1 h at 4 °C. eIF4G-(642-1560) was purified from the S-peptide-tagged NH2-terminal fragment by adsorption of the latter to S-protein-agarose.

Prior to performing binding experiments with S-protein-agarose or m7GTP-Sepharose, purified eIF4G fragments, eIF4A and eIF4F, were passed over desalting Econo-Pac 10 DG disposable chromatography columns to replace the buffer with buffer A plus 5% (v/v) glycerol. Prior to SPR analysis, purified eIF4G fragments and eIF3 were passed over the same columns except they were equilibrated with buffer B (20 mM HEPES-KOH, 150 mM KCl, 2 mM EDTA, 0.05% (v/v) Tween 20, and 0.5 mM beta -mercaptoethanol, pH 7.5). After buffer exchange, the concentrations of proteins were determined using the Bio-Rad protein assay kit, using bovine serum albumin as standard.

Synthesis of [32P]RNA-- RNA corresponding to the EMCV IRES was transcribed in vitro using the Promega Riboprobe system. Briefly, transcription reactions (20 µl) containing BglII-linearized pCite4Gwt (1 µg) (46), 0.5 mM GTP, 0.5 mM CTP, 0.5 mM ATP, 20 µM UTP, 30 µCi of [alpha -32P]UTP, 20 units of T7 RNA polymerase, 10 mM dithiothreitol, and 20 units of RNAsin were incubated at 37 °C for 60 min. Transcription was then terminated by digestion of DNA with RQ1 DNase (1 unit) for 20 min at 30 °C. RNA was extracted with phenol/chloroform and precipitated with ammonium acetate/ethanol prior to storage at -80 °C.

UV Cross-linking of [32P]RNA with eIF4G Fragments-- Several recombinant fragments of eIF4G, eIF4G-(642-1560), S-eIF4G-(613-1078), S-eIF4G-(877-1078), and S-eIF4G-(975-1078) were incubated in the presence of 32P-labeled RNA corresponding to the EMCV IRES. Reactions (17 µl) containing the recombinant eIF4G fragment (4 µg), [32P]RNA (1.5 µCi), RNAsin (10 units), 30 mM HEPES (pH 7.5), 30 mM potassium acetate, 0.6 mM dithiothreitol, and 120 µM spermidine were preincubated for 10 min at 37 °C. Reactions were then spotted onto Parafilm and irradiated on ice in the GS Gene Linker UV chamber (Bio-Rad) for 999 s. Reactions were transferred to Eppendorf tubes and incubated for 15 min at 37 °C in the presence of RNase A (25 µg/ml) and RNase V1 (0.4 units/µl). Samples were analyzed on SDS-PAGE followed by autoradiography.

Protein Binding Assays on m7GTP-Sepharose-- eIF4F was incubated with eIF3 in the presence or absence of the recombinant eIF4G fragments eIF4G-(642-1560), S-eIF4G-(877-1078), or S-eIF4G-(975-1078) for 40 min on ice. Reactions contained at least a 20-fold molar excess of recombinant fragment over intact eIF4G. Proteins were then mixed with m7GTP-Sepharose in the presence of 1% milk proteins in buffer A and incubated for 2 h at 4 °C. Following washing four times with 300-µl aliquots of buffer A, bound material was eluted from the resin with SDS-electrophoresis buffer and analyzed by SDS-PAGE (47), with detection by Coomassie Blue staining.

Protein Binding Assays on S-Protein-Agarose-- Binding of S-eIF4G-(613-1078), S-eIF4G-(877-1078), S-eIF4G-(975-1078), and S-eIF4G-(1078-1560) with eIF3 (and in some cases eIF4A) was performed using S-protein-agarose. After a 40-min preincubation of the S-peptide-tagged eIF4G fragments with eIF3 (and in some cases 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 A containing 1% milk proteins at 4 °C. The resin was washed four times with 200-µl aliquots of buffer A, and the bound proteins were analyzed by SDS-PAGE as described above.

Quantitation of Binding Data-- Quantitation of eIF4G fragments, eIF3 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, run on the same gel, of purified eIF4F, recombinant eIF4G fragments, eIF4A or eIF3 for which the concentrations had been determined with the Bio-Rad protein assay kit. Curve fitting was performed using SigmaPlot software version 4.01 (SPSS, Inc.). In cases of eIF3 binding with S-eIF4G-(613-1078) or S-eIF4G-(975-1078), the data were fit with an equation describing the Langmuir isotherm:


BR=n[<UP>eIF3</UP>]<SUB><UP>f</UP></SUB><UP>/</UP>(K<SUB>d</SUB>+[<UP>eIF3</UP>]<SUB><UP>f</UP></SUB>) (Eq. 1)
where BR is the binding ratio, i.e. the molar ratio of bound eIF3 to the eIF4G fragment, n is the number of eIF3-binding sites on the eIF4G fragment, [eIF3]f is the concentration of eIF3 not bound to the resin, and Kd is the dissociation equilibrium constant for the eIF3·eIF4G complex. A non-linear least squares fit was performed in which n and Kd were allowed to vary. The number of binding sites was verified by replotting the data according to Scatchard (48).

In the case of eIF3 binding to eIF4G in the presence or absence of eIF4A, Eq. 1 was used assuming that n = 1 for eIF3, even when saturation had not been achieved. The 1:1 stoichiometry for binding of S-eIF4G-(613-1078) to eIF3 in the presence of eIF4A was confirmed in experiments using higher concentrations of components, when saturation of all binding sites was achieved (data not shown).

In the case of eIF4A binding to S-eIF4G-(613-1078) or S-eIF4G-(1078-1560) in the presence or absence of eIF3, Eq. 2 was used assuming that n = 1 for eIF4A in each eIF4G fragment, even when saturation was not achieved:
BR=n[<UP>eIF4A</UP>]<SUB><UP>f</UP></SUB><UP>/</UP>(K<SUB>d</SUB>+[<UP>eIF4A</UP>]<SUB><UP>f</UP></SUB>) (Eq. 2)
The 1:1 stoichiometry for eIF4A binding to each eIF4G fragment in the presence of eIF3 was similarly confirmed in experiments using saturating concentrations of factors (data not shown).

SPR Analysis of eIF4G·eIF3 Interactions-- SPR was carried out using BIAcore 2000 instrument (BIAcore, Inc., Piscataway, NJ). eIF3 was immobilized on a research grade CM5 sensor chip using the amino-coupling kit supplied by the manufacturer in 10 mM sodium acetate, pH 3.5. The surface density of immobilized eIF3 was 1500-1800 RU. One RU corresponds to an immobilized protein density of 1 pg/mm2 (49). 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 eIF3-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 B at 25 °C and a flow rate of 20 µl/min. At least six different concentrations of each eIF4G fragment were injected for each experiment. The first injection contained buffer without the eIF4G fragment. Between injections, the surface was regenerated with buffer C (20 mM HEPES-KOH, 500 mM KCl, 3 mM EDTA, 0.1% (v/v) Tween 20, 2 mM beta -mercaptoethanol, pH 7.5) at a flow rate of 40 µl/min and contact time of 3 min, followed by buffer B 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 iff  AB) as described elsewhere (42). Values for the statistical closeness of fit, chi 2, were always below 10, indicating that the simple 1:1 model of interaction correctly described the experimental data.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Delineation of the Minimal eIF3-binding Site on eIF4G-- An earlier study indicated that eIF3 binds to the region of human eIF4G from aa 635 to 1041, which constitutes the central domain (24). This was confirmed in subsequent studies with eIF4G fragments representing aa 613-1090 (31) and aa 672-1065 (41). To characterize the eIF3-binding site further, S-eIF4G fragments of decreasing size (Fig. 1A) were incubated with eIF3 and then immobilized on S-protein-agarose. eIF3 was specifically retained through binding to S-eIF4G-(613-1078), S-eIF4G-(877-1078), or S-eIF4G-(975-1078) (Fig. 1B, lanes 2-4, respectively). Control experiments in which the S-eIF4G fragment was omitted (lane 1) or was replaced with S-eIF4G-(1078-1560), a fragment that does not contain the eIF3-binding site, indicated no binding (data not shown). Thus, the smallest fragment that binds eIF3 is S-eIF4G-(975-1078).


View larger version (28K):
[in this window]
[in a new window]
  		Fig. 1.   A, schematic representation of human eIF4G-1 and recombinant fragments. The binding sites for various initiation factors and RNA determined in this and other studies are shown in shaded boxes, with aa numbers located below delineating the borders. The arrow labeled 2A pro indicates the site of entero- and rhinoviral 2A protease cleavage (39). Various recombinant proteins containing the indicated portions of eIF4G are shown below, with inclusive aa numbers. B, binding of eIF3 to eIF4G fragments. eIF3 was incubated with S-eIF4G-(613-1078) (lane 2), S-eIF4G-(877-1078) (lane 3), S-eIF4G-(975-1078) (lane 4), or without any eIF4G fragments (lane 1) and then captured on S-protein-agarose. Material bound to the resin was eluted and subjected to SDS-PAGE and Coomassie Blue staining.

S-eIF4G-(975-1078) Competes with Intact eIF4G for Binding with eIF3-- It was conceivable that eIF4G contained eIF3 binding determinants in addition to those in S-eIF4G-(975-1078). If so, S-eIF4G-(975-1078) would not be expected to compete for the binding of eIF3 to full-length eIF4G. To test this, we incubated rabbit reticulocyte eIF4F and eIF3 in the presence or absence of various recombinant eIF4G fragments. The mixture was then fractionated on m7GTP-Sepharose, the eIF4F complex (with bound eIF3) being retained by virtue of the eIF4E component (Fig. 2). (The competitor recombinant eIF4G fragments did not contain the eIF4E-binding site; see Fig. 1A.) Control reactions did not contain any recombinant eIF4G fragments (lanes 4, 8, and 12). All three fragments, eIF4G-(642-1560), S-eIF4G-(877-1078), and S-eIF4G-(975-1078), competed with intact eIF4F for binding to eIF3. In the presence of the recombinant eIF4G fragments, retention of eIF3 on m7GTP-Sepharose was reduced but not that of eIF4G and eIF4E3 (Fig. 2, cf. lane 12 with lanes 9-11). Notably, even the smallest eIF4G fragment, containing only aa 975-1078, competed with intact rabbit eIF4F for binding to eIF3.


View larger version (70K):
[in this window]
[in a new window]
  		Fig. 2.   Recombinant human eIF4G fragments compete with intact rabbit eIF4F for association with eIF3. eIF4F was incubated with eIF3 either in the absence (lanes 4, 8, and 12) or presence of S-eIF4G-(975-1078) (lanes 1, 5, and 9), S-eIF4G-(877-1078) (lanes 2, 6, and 10), or S-eIF4G-(642-1560) (lanes 3, 7, and 11). The mixtures were then fractionated on m7GTP-Sepharose. Aliquots from the starting reaction mixtures (lanes 1-4), column flow-through (lanes 5-8), and material eluted with SDS-electrophoresis buffer (lanes 9-12) were subjected to SDS-PAGE on a 12% gel followed by staining with Coomassie Blue. The position of intact eIF4G, eIF4E, and the recombinant eIF4G fragments are indicated. Asterisks show the positions of eIF3 subunits.

The RNA-binding Site on eIF4G Is Distinct from the eIF3-binding Site-- The central domain of eIF4G binds both eIF3 (24) and the EMCV IRES (38, 37). This suggests that eIF3 may associate indirectly with eIF4G through an RNA bridge, because eIF3 also binds RNA (see the introduction). To test this hypothesis, we determined the site of RNA binding on eIF4G by UV cross-linking. Various eIF4G fragments were incubated with 32P-labeled RNA representing the EMCV IRES. eIF4G-(642-1560) and S-eIF4G-(613-1078) were found to cross-link to RNA (Fig. 3B, lanes 5 and 6, respectively), whereas S-eIF4G-(877-1078) and S-eIF4G-(975-1078) were not (lanes 7 and 8, respectively). This suggests that the RNA-binding site (or an essential portion of it) is located between aa 642 and 876. This region overlaps with the central eIF4A-binding site (31, 41, 42) but is distinct from the eIF3-binding site (Fig. 1B). Thus, eIF4G binds eIF3 directly, not through an RNA bridge.


View larger version (59K):
[in this window]
[in a new window]
  		Fig. 3.   The RNA-binding site is located between aa 613 and 876 and is therefore distinct from the eIF3-binding site. Recombinant eIF4G fragments were incubated in the presence of 32P-labeled RNA representing the EMCV IRES, cross-linked to RNA using UV light, digested with a mixture of RNases, and subjected to SDS-PAGE on 10% gel followed by staining with Coomassie Blue (A) and autoradiography (B). Unreacted proteins (lanes 1-4) and UV cross-linked proteins (lanes 5-8) correspond to eIF4G-(642-1560) (lanes 1 and 5), S-eIF4G-(613-1078) (lanes 4 and 8), S-eIF4G-(877-1078) (lanes 3 and 7), and S-eIF4G-(975-1078) (lanes 4 and 8).

Another approach to answer the same question was to incubate eIF3 with S-eIF4G-(613-1078) in the presence of micrococcal nuclease and then subject the mixture to S-protein-agarose, as in Fig. 1B. The results indicated that eIF3 was bound equally, regardless of the presence of micrococcal nuclease (data not shown), confirming that there is no RNA bridge.

S-eIF4G-(613-1078) and S-eIF4G-(975-1078) Bind eIF3 with a Stoichiometry of 1:1-- To estimate the stoichiometry for the eIF4G·eIF3 interaction, we carried out titration experiments and then used S-protein-agarose to capture the complex. S-eIF4G-(613-1078) was incubated with either no eIF3 or a range of concentrations varying from a 0.5- to 10-fold molar ratio. The S-eIF4G-(613-1078)·eIF3 complex was adsorbed to S-protein-agarose, eluted, and analyzed by SDS-PAGE with Coomassie Blue staining (Fig. 4A). The binding data were quantitated using standard curves derived from eIF3 and the eIF4G fragment run on the same gel. A non-linear least-squares fit of the experimental data was performed using Eq. 1 (Fig. 4B). The results indicated that the number of eIF3-binding sites on S-eIF4G-(613-1078), n, was 1.3 ± 0.1. A linear transform of the data indicated the existence of a single-binding site (Fig. 4B, inset).


View larger version (57K):
[in this window]
[in a new window]
  		Fig. 4.   Saturation analysis of eIF3 binding to eIF4G fragments. A, S-eIF4G-(613-1078) was incubated at a concentration of 0.02 µM with eIF3 at 0, 0.01, 0.016, 0.024, 0.04, 0.1, and 0.2 µM (lanes 1-7, respectively). The S-eIF4G-(613-1078) and bound eIF3 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. In lane 8, 0.2 µM eIF3 was incubated with S-protein-agarose in the absence of any eIF4G fragment. B, the amounts of S-eIF4G-(613-1078) and eIF3 in A were quantitated 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 eIF3 to S-eIF4G-(613-1078), and [eIF3]f is the total eIF3 concentration minus the complexed eIF3. The curve is a least squares fit of Eq. 1 to the data, in which n and Kd are allowed to vary (see "Experimental Procedures"). The curve shown corresponds to n = 1.3 ± 0.1. The coefficient of determination (R2) was 0.99. Inset, replot of the data as BR/[eIF3]f versus BR. C, same as A except that S-eIF4G-(975-1078) was used at 0.1 µM, and the eIF3 concentrations were 0, 0.05, 0.1, 0.25, 0.45, 0.65, 0.8, and 1 µM for lanes 1-8, respectively. Lane 9, 1 µM eIF3 was incubated with S-protein-agarose in absence of any eIF4G fragment. D, quantitation of the data in C by the same method as described in B. The curve shown corresponds to n = 1.1 ± 0.1. The R2 value was 0.99. Inset, the data of D are replotted as in B, inset.

A similar experiment was performed with S-eIF4G-(975-1078). The eIF4G fragment was incubated with a range of eIF3 concentrations from a 0.5- to 10-fold molar ratio (Fig. 4C). The data were quantitated as described above. The non-linear least-squares fit yielded n = 1.1 ± 0.1 (Fig. 4D). Scatchard analysis confirmed the existence of a single-binding site (Fig. 4D, inset). These experiments indicate that, within experimental error, the stoichiometries of eIF3 binding to both S-eIF4G-(613-1078) and S-eIF4G-(975-1078) were 1:1.

Kinetic Measurements of eIF3-eIF4G Fragment Interactions Using SPR-- The S-protein-agarose method of analysis does not yield accurate Kd values because it represents a non-equilibrium situation; the eIF3 concentration during the washes is much lower than during the initial incubation, giving an overestimate for the Kd. We therefore turned to SPR, a more quantitative method, to measure both affinities and rate constants for binding of eIF4G fragments to eIF3.

eIF3 was immobilized on the sensor chip by the amino-coupling procedure, and eIF4G-(642-1560), S-eIF4G-(613-1078), or S-eIF4G-(975-1078) fragments were passed over it (Fig. 5). As a control, a protein containing the same NH2-terminal tags (thioredoxin, His6, and S-peptide) as the S-eIF4G fragments but no eIF4G sequences was passed over the immobilized eIF3. The results indicated no binding of the control protein to eIF3 (data not shown).


View larger version (29K):
[in this window]
[in a new window]
  		Fig. 5.   Kinetic analysis of eIF4G-(642-1560), S-eIF4G-(613-1078), and S-eIF4G-(975-1078) binding to eIF3 by SPR. A, buffer B containing either 0, 3, 10, 25, 50, 100, or 200 nM eIF4G-(642-1560) was passed over eIF3 immobilized on a sensor chip (successive curves from lowest to highest). 540 s after injection, the mobile phase was changed back to buffer B alone. Thin traces correspond to a global fit of the data with a 1:1 binding model. The sensor chip contained 1800 RU of immobilized eIF3. B, the same as A except that 0, 3, 10, 25, 50, 100, or 200 nM solutions of S-eIF4G-(613-1078) were passed over the same sensor chip containing 1800 RU of eIF3. C, S-eIF4G-(975-1078) at concentrations of 0, 3, 10, 25, 50, 100, and 200 nM was passed over a sensor chip containing 1500 RU of eIF3 as in A.

The experimental data for binding of eIF4G-(642-1560), S-eIF4G-(613-1078), and S-eIF4G-(975-1078) to eIF3 were fit to the simple 1:1 binding model (thin lines in Fig. 5 (A-C); see "Experimental Procedures"). The low values for the statistical closeness of fit, chi 2, and for the maximal residuals, which represent deviation of actual data from theoretical, indicate that the 1:1 binding model provides a good fit to the data (Table I). The rate constants for association (ka), rate constants for dissociation (kd), and equilibrium dissociation constants (Kd = kd/ka) are also shown in Table I. The results show that all three eIF4G fragments had similar kinetic and equilibrium characteristics for binding to eIF3, suggesting that, within experimental error, the S-eIF4G-(975-1078) fragment contains the entire eIF3-binding site.

                              
View this table:
[in this window]
[in a new window]
 
Table I
Kinetic and affinity constants for interactions between recombinant eIF4G fragments and immobilized eIF3
Kinetic constants were derived from several independent SPR experiments, similar to Fig. 5, on a sensor chip with eIF3 immobilized by amino-coupling. Seven different concentrations of the indicated recombinant eIF4G fragments, ranging from 0 to 200 nM, were used in each experiment. The apparent dissociation constants (Kd) for the interactions were calculated from kd/ka. chi 2 represents the statistical closeness of fit. Residuals represent the maximal deviation of experimental data from the theoretical curve. Values are reported ± S.E. In the case of eIF4G-(642-1560), S.E. was derived from a single sensor chip probed with six different concentrations of the eIF4G fragment. For S-eIF4G-(613-1078), five independent experiments were performed and the mean and S.E. calculated from them. For S-eIF4G-(975-1078), the results are based on three independent experiments.

eIF4A Enhances the Binding of eIF3 to S-eIF4G-(613-1078)-- To explore the possible influence of eIF4A on the binding of eIF3 to the central region of eIF4G, two types of titration experiments were carried out. In the first, S-eIF4G-(613-1078) was incubated with a fixed amount of eIF3 and varying amounts of eIF4A. The bound proteins were then analyzed as in Fig. 4 (Fig. 6A). The molar ratio of bound eIF3 to S-eIF4G-(613-1078) for each reaction was plotted against the concentration of eIF4A present in each reaction (Fig. 6B). The results indicated that, at higher concentrations of eIF4A, more eIF3 was retained on the resin through the interaction with S-eIF4G-(613-1078). eIF4A enhanced the binding of eIF3 to S-eIF4G-(613-1078) by more than 4-fold over the range of concentrations tested.


View larger version (56K):
[in this window]
[in a new window]
  		Fig. 6.   eIF4A enhances the binding of eIF3 to S-eIF4G-(613-1078). A, S-eIF4G-(613-1078) was incubated at a concentration of 0.6 µM with a 0.5 µM eIF3 and either no eIF4A (lane 1) or 0.34, 0.7, 1.2, 3, and 6 µM eIF4A (lanes 2-6, respectively) and then fractionated on S-protein-agarose. As a control, 6 µM eIF4A and 0.5 µM eIF3 were incubated with S-protein-agarose in the absence of an eIF4G fragment (lane 7). Material bound to the resin was subjected to SDS-PAGE on a 10% gel followed by staining with Coomassie Blue. B, the amounts of S-eIF4G-(613-1078) and eIF3 in A were quantitated by scanning stained bands and comparing the signals to standard curves of the two purified proteins electrophoresed on the same gel (not shown). The binding ratio is the molar ratio of eIF3 to S-eIF4G-(613-1078), and [eIF4A] is the total eIF4A concentration present in the reaction. C, S-eIF4G-(613-1078) was incubated at a concentration of 0.05 µM with varying amounts of eIF3 (0.04, 0.075, 0.15, 0.2, 0.5 µM) in the presence (lanes 6-10) or absence (lanes 1-5) of 0.1 µM eIF4A and fractionated on S-protein-agarose. Material bound to the resin was subjected to SDS-PAGE on a 7% gel followed by staining with Coomassie Blue. D, the binding data in C in the presence () or absence of (black-triangle) eIF4A to were fit to Eq. 1 by least squares minimization in which Kd was allowed to vary and assuming that n = 1 in both cases. The R2 values were 0.91 (-eIF4A) and 0.96 (+eIF4A).

The effect of eIF4A on the affinity of eIF3 for eIF4G could be due to an eIF4A-induced change in the structure of either eIF4G or eIF3. To distinguish between these two possibilities, we used an eIF4G fragment that had an eIF3-binding site but no eIF4A-binding site, S-eIF4G-(975-1078). We incubated this fragment with eIF3 and [14C]eIF4A and then subjected the mixture to S-protein-agarose. eIF3 was efficiently retained on the resin through S-eIF4G-(975-1078), but there was no detectable retention of [14C]eIF4A, as determined by autoradiography (data not shown). This confirmed that eIF3 does not bind to eIF4A directly. It also rules out the possibility that eIF4A binds to eIF3 only after the latter is bound to eIF4G, because the eIF3·eIF4G complex retained by S-protein-agarose contained no eIF4A. Thus, the contrary hypothesis is supported, that the increase in eIF3 binding to S-eIF4G-(613-1078) upon addition of eIF4A (Fig. 6B) is due to an eIF4A-induced change in eIF4G.

The second type of experiment was titration of S-eIF4G-(613-1078) with eIF3 in presence or absence of a fixed concentration of eIF4A (Fig. 6, C and D). The presence of 0.1 µM eIF4A in the reaction mixture enhanced the amount of eIF3 bound to S-eIF4G-(613-1078) by 2- to 3-fold at low concentrations of eIF3, but the enhancement decreased as the eIF3-binding site became saturated.

Because there are two independent variables involved in studying the influence of one ligand on binding of another ligand (viz., eIF3 concentration and eIF4A concentration), it is necessary to fix one arbitrarily while varying the other. These two experiments do not explore eIF3·eIF4G binding over the full range of possible eIF4A and eIF3 concentrations. Nonetheless, both types of experiments show that eIF4A increases eIF3 binding to the central domain of eIF4G, probably through a conformational change in eIF4G structure.

eIF3 Enhances the Binding of eIF4A to S-eIF4G-(613-1078), but Not the Binding of eIF4A to S-eIF4G-(1078-1560)-- To study the reciprocal relationship, viz., the effect of eIF3 on binding of eIF4A to eIF4G, we performed titration experiments using two different eIF4G fragments. One represented the central domain, S-eIF4G-(613-1078), and therefore contained both eIF4A- and eIF3-binding sites. The other represented the COOH-terminal domain, S-eIF4G-(1078-1560), and therefore contained only an eIF4A-binding site. Thus, if eIF3 affects the interaction between eIF4A and eIF4G by changing the conformation of the latter, one would expect to see a difference with S-eIF4G-(613-1078) but not S-eIF4G-(1078-1560).

S-eIF4G-(613-1078) and S-eIF4G-(1078-1560) were incubated separately with varying amounts of [14C]eIF4A in the presence or absence of eIF3. Proteins bound to eIF4G were retained on S-protein-agarose. The amounts of the eIF4G fragment and eIF3 bound to the resin were analyzed by SDS-PAGE and Coomassie Blue staining (Fig. 7, A and D), whereas the amounts of [14C]eIF4A bound were analyzed by autoradiography (Fig. 7, B and E). Quantitative analysis was performed by fitting the experimental data to Eq. 2, assuming that each eIF4G fragment has one eIF4A-binding site (Fig. 7, C and F). This assumption has been independently validated (42). The results indicated that the presence of 0.1 µM eIF3 in the reaction mixture increased the binding of eIF4A to the central domain (decreased the Kd) by 2.4-fold (Fig. 7C) but did not alter the binding of eIF4A to the COOH-terminal domain (Fig. 7F).


View larger version (45K):
[in this window]
[in a new window]
  		Fig. 7.   Effect of eIF3 on binding of eIF4A to S-eIF4G-(613-1078) and S-eIF4G-(1078-1560). A, S-eIF4G-(613-1078) (0.1 µM) was incubated with varying amount of eIF4A (0.1, 0.15, 0.2, and 0.35 µM) in the absence (lanes 1-4) or presence (lanes 5-8) of 0.1 µM eIF3 and then fractionated on S-protein-agarose. In lane 9, S-eIF4G-(613-1078) was incubated with eIF3 in the absence of eIF4A. Material bound to the resin was subjected to SDS-PAGE on an 8% gel followed by staining with Coomassie Blue. B, autoradiography of [14C]eIF4A bound to the resin through interaction with S-eIF4G-(613-1078). C, quantitation of binding data from A and B. The data were fit to Eq. 2 (see "Experimental Procedures") by non-linear least squares minimization, assuming that n = 1. The R2 values for the fits in the absence (black-triangle) and presence () of eIF3 were 0.67 and 0.99, respectively. D, S-eIF4G-(1078-1560) (0.1 µM) was incubated with varying amounts of eIF4A (0.1, 0.15, 0.2, and 0.35 µM) in the absence (lanes 1-4) or presence (lanes 5-8) of 0.1 µM eIF3 and then fractionated as in A. In lane 9, S-eIF4G-(1078-1560) was incubated with eIF3 in the absence of eIF4A. E, autoradiography of [14C]eIF4A bound to the resin from D. F, quantitation of binding data from D and E, as described in C. The R2 values in the absence (black-triangle) or presence () of eIF3 were 0.97 and 0.86, respectively.

Differences in eIF4A binding to eIF4G in the presence versus absence of eIF3 are more pronounced at low eIF4A concentrations, when the eIF4A-binding site is not saturated. However, it was necessary to show that the stoichiometry of eIF4A to the central domain of eIF4G remained 1:1 in the presence of eIF3, which requires high concentrations of eIF4A. Therefore, we performed an experiment similar to that shown in Fig. 7C except that the maximum concentration of eIF4A was 4 µM and the fixed concentration of S-eIF4G-(613-178) was 0.5 µM. The results indicated that, within experimental error, the stoichiometry of eIF4A binding to eIF4G was still 1:1 (data not shown).

Finally, we tested natural rabbit reticulocyte eIF4F for cooperativity of eIF4A and eIF3 binding to rule out artifacts due to the use of recombinant proteins. eIF4F (0.02 µM) was incubated with 0.05 µM eIF4A in the presence or absence of 0.03 µM eIF3. Bound proteins were captured on m7GTP-Sepharose. The results indicated that eIF3 increased the binding of eIF4A to eIF4F by 1.6-fold (data not shown). Thus, eIF3 stimulates the binding of eIF4A to full-length eIF4G present in the eIF4F complex.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The finding that the eIF3-binding site on eIF4G is located between aa 975 and 1078 (Fig. 1B), combined with a recent study showing that it is located between aa 672 and 1065 (41), indicates that it is actually between aa 975 and 1065, although this specific fragment has not been tested directly. Despite the fact that this is a rather small portion (104 aa) of the 1560-aa eIF4G molecule, the similarity of Kd values for eIF4G-(642-1560), eIF4G-(613-1078), and eIF4G-(975-1078) (Table I) suggests that all of the binding determinants for eIF3 are located within this region. This places the eIF3-binding site between the two eIF4A-binding sites with no overlap (Fig. 1A). Similarly, there is no overlap between the RNA-binding site and the eIF3-binding site, because S-eIF4G-(975-1078) does not bind RNA (Fig. 3B). The latter finding indicates that the interaction between eIF4G and eIF3 does not require RNA.

Contrasting with the model of eIF4G with widely separated, non-overlapping binding sites (Fig. 1A) is the finding that binding of some ligands to eIF4G positively influence the binding of others. In the current work, we showed that eIF4A enhances the binding of eIF3 to eIF4G (Fig. 6) and, reciprocally, that eIF3 enhances the binding of eIF4A to the central (but not COOH-terminal) domain of eIF4G (Fig. 7). In other studies, it was shown that binding of eIF4A to eIF4G fragments containing both the central and COOH-terminal eIF4A-binding sites was greater than the sum of binding to fragments containing the individual sites, suggesting positive cooperativity between the two eIF4A-binding sites (42). There may also be cooperative binding of eIF4B and RNA to eIF4G; mammalian eIF4B increases binding of eIF4G-(613-1088) to RNA (38). Also, wheat germ eIF(iso)4G in complex with eIF(iso)4E binds PABP better than eIF(iso)4G alone (30), suggesting that eIF4E and PABP bind eIF4G cooperatively. Titration experiments were not performed in these published studies involving binding of RNA, eIF4B, PABP, and eIF4E to eIF4G, so we can only speculate that there is a cooperative change in the respective Kd values. However, titration experiments were performed in the case of eIF4A (Fig. 7C) and eIF3 (Fig. 6D), allowing us to draw this conclusion. This paints a picture of eIF4G as a dynamic motor facilitating the stepwise interaction of initiation factors, ribosomes, and mRNA rather than as a static scaffold for assembly of the initiation complex. The cooperativity likely involves a conformational change in eIF4G, because eIF4A and eIF3 do not appear to interact directly.

Several previous results appear at first to be incompatible with the existence of non-overlapping eIF4A- and eIF3-binding sites. eIF4G variants in which the central eIF4A-binding site is changed by site-directed mutagenesis also fail to bind eIF3 in cell extracts (31). eIF4G fragments with truncations extending into the central eIF4A-binding site but containing an intact eIF3-binding site (eIF4G-(702-1090), eIF4G-(733-1090), and eIF4G-(762-1090)) lose not only eIF4A binding but also eIF3 binding (41). Conversely, eIF4G fragments containing an intact central eIF4A-binding site but lacking the eIF3-binding site (eIF4G-(642-947) (Ref. 41) and eIF4G-(642-877) (Ref. 40)) fail to bind not only eIF3 but also eIF4A. The findings reported in the present study may resolve these apparent contradictions: positive cooperativity between eIF4A and eIF3 predicts that the absence of binding of one ligand would weaken binding of the other ligand.

Our studies indicate that the Kd for binding of mammalian eIF3 to eIF4G is about 42 nM in the absence of eIF4A (Table I). The Kd for the wheat germ eIF3-eIF(iso)4F interaction was found to be 460 nM in the absence of salt and 1.28 µM in the presence of 100 mM KCl and 2 mM MgCl2 (50), or about 10- to 32-fold weaker. Interestingly, the eIF4F and eIF(iso)4F complexes from wheat germ do not contain eIF4A (51-53), suggesting that the affinity of eIF4A for the central domain of wheat germ eIF4G is also weaker than for mammalian eIF4G. Furthermore, plant eIF4G does not contain the second eIF4A-binding site in the COOH terminus, which acts cooperatively to stimulate binding of eIF4A to the central site (42) and recruitment of mRNA to the ribosome (41). Both the lower affinity of eIF3 for eIF4G and the absence of a COOH-terminal eIF4A-binding site would lead to a weaker binding of eIF4A to the central domain, according to the finding of positive cooperativity involving both sites (Fig. 7 and Ref. 42). The weaker affinity makes the loss of eIF4A from plant eIF4F more likely than from mammalian eIF4F.

It is also interesting to note that, although the COOH-terminal domain of eIF4G serves to stimulate eIF4A binding (42) and enhance function of the central domain in protein synthesis both in vivo (40) and in vitro (41), it does not influence eIF3 binding. As shown in Fig. 5, the 918-aa eIF4G fragment S-eIF4G-(642-1560), which contains both central and COOH-terminal domains, binds eIF3 with the same affinity as the 103-aa fragment S-eIF4G-(975-1078), which contains only a small portion of the central domain. Thus, the COOH-terminal domain is somehow able to affect binding of the distal, 46-kDa eIF4A molecule without affecting binding of the proximal, 600-kDa eIF3 molecule.

    ACKNOWLEDGEMENTS

We thank William C. Merrick for providing [14C]eIF4A, Sherry Long for help with analysis of SPR data, and Aili Cai for valuable technical assistance.

    FOOTNOTES

* This work was supported by Grant GM20818 from the National Institute of General Medical Sciences.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.

Dagger 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 Ctr., 1501 Kings Highway, Shreveport, LA 71130-3932. Tel.: 318-675-5161; Fax: 318-675-5180; E-mail: rrhoad@lsuhsc.edu.

Published, JBC Papers in Press, October 5, 2000, DOI 10.1074/jbc.M007525200

2 These two eIF3 subunits migrate at the same position under most SDS-PAGE conditions; it is unknown which of them binds RNA.

3 Detection of eIF4A is difficult in the presence of eIF3, because the p44, p47, and p48 subunits of eIF3 co-migrate similarly to eIF4A. Furthermore, one of the competitors, eIF4G-(642-1560), contains an eIF4A-binding site and would be expected to compete with eIF4F for any eIF4A in the reaction mixture.

    ABBREVIATIONS

The abbreviations used are: Met-tRNAi, methionyl initiator tRNA; eIF, eukaryotic initiation factor; aa, amino acid residue; BR, molar binding ratio of eIF4A or eIF3 to eIF4G fragments; EMCV IRES, encephalomyocarditis virus internal ribosomal entry site; PABP, poly(A)-binding protein; PAGE, polyacrylamide gel electrophoresis; RU, response unit(s); SPR, surface plasmon resonance.

    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 Press, Cold Spring Harbor, NY
2. Pestova, T. V., Lomakin, I. B., Lee, J. H., Choi, S. K., Dever, T. E., and Hellen, C. U. T. (2000) Nature 403, 332-335
3. Pestova, T. V., Borukhov, S. I., and Hellen, C. U. T. (1998) Nature 394, 854-859
4. Benne, R., and Hershey, J. W. B. (1978) J. Biol. Chem. 253, 3078-3087
5. Trachsel, H., and Staehelin, T. (1979) Biochim. Biophys. Acta 565, 305-315
6. Chaudhuri, J., Chowdhury, D., and Maitra, U. (1999) J. Biol. Chem. 274, 17975-17980
7. Hershey, J. W. B., Asano, K., Naranda, T., Vornlocher, H. P., Hanachi, P., and Merrick, W. C. (1996) Biochimie (Paris) 78, 903-907
8. Asano, K., Kinzy, T. G., Merrick, W. C., and Hershey, J. W. (1997) J. Biol. Chem. 272, 1101-1109
9. Phan, L., Zhang, X., Asano, K., Anderson, J., Vornlocher, H.-P., Greenberg, J. R., Qin, J., and Hinnebusch, A. G. (1998) Mol. Cell. Biol. 18, 4935-4946
10. Brown-Luedi, M. L., Meyer, L. J., Milburn, S. C., Yau, P. M. P., Corbett, S., and Hershey, J. W. B. (1982) Biochemistry 21, 4202-4206
11. Westermann, P., and Nygard, O. (1984) Nucleic Acids Res. 12, 8887-8897
12. Setyono, B., Van Steeg, H., and Voorma, H. O. (1984) Biochim. Biophys. Acta 782, 242-246
13. Westermann, P., Sohi, M. K., and Arnstein, H. R. V. (1986) FEBS Lett. 205, 171-174
14. Sizova, D. V., Kolupaeva, V. G., Pestova, T. V., Shatsky, I. N., and Hellen, C. U. (1998) J. Virol. 72, 4775-4782
15. Block, K. L., Vornlocher, H. P., and Hershey, J. W. (1998) J. Biol. Chem. 273, 31901-31908
16. Asano, K., Vornlocher, H.-P., Richter-Cook, N. J., Merrick, W. C., Hinnebusch, A. G., and Hershey, J. W. B. (1997) J. Biol. Chem. 272, 27042-27052
17. Oderman-Macchioli, F. E., Tisminetzky, S. G., Zotti, M., Baralle, F. E., and Buratti, E. (2000) Nucleic Acids Res. 28, 875-885
18. Nygard, O., and Westermann, P. (1982) Nucleic Acids Res. 10, 1327-1334
19. Tolan, D. R., Hershey, J. W., and Traut, R. T. (1983) Biochimie (Paris) 65, 427-436
20. Fletcher, C. M., Pestova, T. V., Hellen, C. U., and Wagner, G. (1999) EMBO J. 18, 2631-2637
21. Methot, N., Song, M. S., and Sonenberg, N. (1996) Mol. Cell. Biol. 16, 5328-5334
22. Bandyopadhyay, A., and Maitra, U. (1999) Nucleic Acids Res. 27, 1331-1337
23. Vornlocher, H. P., Hanachi, P., Ribeiro, S., and Hershey, J. W. (1999) J. Biol. Chem. 274, 16802-16812
24. Lamphear, B. J., Kirchweger, R., Skern, T., and Rhoads, R. E. (1995) J. Biol. Chem. 270, 21975-21983
25. Keiper, B. D., Gan, W., and Rhoads, R. E. (1999) Int. J. Biochem. Cell Biol. 31, 37-41
26. Mader, S., Lee, H., Pause, A., and Sonenberg, N. (1995) Mol. Cell. Biol. 15, 4990-4997
27. Metz, A. M., and Browning, K. (1996) J. Biol. Chem. 271, 31033-31036
28. Imataka, H., Gradi, A., and Sonenberg, N. (1998) EMBO J. 17, 7480-7489
29. Tarun, S. Z., and Sachs, A. B. (1996) EMBO J. 15, 7168-7177
30. Le, H., Tanguay, R. L., Balasta, M. L., Wei, C. C., Browning, K., Metz, A. M., Goss, D. J., and Gallie, D. R. (1997) J. Biol. Chem. 272, 16247-16255
31. Imataka, H., and Sonenberg, N. (1997) Mol. Cell. Biol. 17, 6940-6947
32. Neff, C. L., and Sachs, A. B. (1999) Mol. Cell. Biol. 19, 5557-5564
33. Dominguez, D., Altmann, M., Benz, J., Baumann, U., and Trachsel, H. (1999) J. Biol. Chem. 274, 26720-26726
34. Pyronnet, S., Imataka, H., Gingras, A.-C., Fukunaga, R., Hunter, T., and Sonenberg, N. (1999) EMBO J. 18, 270-279
35. Waskiewicz, A. J., Johnson, J. C., Penn, B., Mahalingam, M., Kimball, S. R., and Cooper, J. A. (1999) Mol. Cell. Biol. 19, 1871-1880
36. Goyer, C., Altmann, M., Lee, H. S., Blanc, A., Deshmukh, M., Woolford, J. L., Jr., Trachsel, H., and Sonenberg, N. (1993) Mol. Cell. Biol. 13, 4860-4874
37. Kolupaeva, V. G., Pestova, T. V., Hellen, C., and Shatsky, I. N. (1998) J. Biol. Chem. 273, 18599-18604
38. Pestova, T. V., Shatsky, I. N., and Hellen, C. U. (1996) Mol. Cell. Biol. 16, 6870-6878
39. Lamphear, B. J., Yan, R., Yang, F., Waters, D., Liebig, H.-D., Klump, H., Kuechler, E., Skern, T., and Rhoads, R. E. (1993) J. Biol. Chem. 268, 19200-19203
40. De Gregorio, E., Preiss, T., and Hentze, M. W. (1999) EMBO J. 18, 4865-4874
41. Morino, S., Imataka, H., Svitkin, Y. V., Pestova, T. V., and Sonenberg, N. (2000) Mol. Cell. Biol. 20, 468-477
42. Korneeva, N. L., Lamphear, B. J., Hennigan, F. L. C., Merrick, W. C., and Rhoads, R. E. (2001) J. Biol. Chem. 276, in press
43. Yoder-Hill, J., Pause, A., Sonenberg, N., and Merrick, W. C. (1993) J. Biol. Chem. 268, 5566-5573
44. Lamphear, B. J., and Panniers, R. (1990) J. Biol. Chem. 265, 5333-5336
45. Liebig, H.-D., Ziegler, E., Yan, R., Hartmuth, K., Klump, H., Kowalski, H., Blaas, D., Sommergruber, W., Frasel, L., Lamphear, B., Rhoads, R. E., Kuechler, E., and Skern, T. (1993) Biochemistry 32, 7581-7588
46. Lamphear, B. J., and Rhoads, R. E. (1996) Biochemistry 35, 15726-15733
47. Laemmli, U. K. (1970) Nature 227, 680-685
48. Scatchard, G. (1949) Ann. N. Y. Acad. Sci. 51, 600-672
49. Stenberg, E., Persson, B., Roos, H., and Urbaniczky, C. (1991) J. Colloid Interface Sci. 143, 513-526
50. Carberry, S. E., and Goss, D. J. (1991) Biochemistry 30, 6977-6982
51. Lax, S., Fritz, W., Browning, K., and Ravel, J. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 330-333
52. Seal, S. N., Schmidt, A., Marcus, A., Edery, I., and Sonenberg, N. (1986) Arch. Biochem. Biophys. 246, 710-715
53. Browning, K. S., Lax, S. R., and Ravel, J. M. (1987) J. Biol. Chem. 262, 11228-11232

Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 Mol. Cell. ProteomicsHome page
E. Damoc, C. S. Fraser, M. Zhou, H. Videler, G. L. Mayeur, J. W. B. Hershey, J. A. Doudna, C. V. Robinson, and J. A. Leary
Structural Characterization of the Human Eukaryotic Initiation Factor 3 Protein Complex by Mass Spectrometry
Mol. Cell. Proteomics, July 1, 2007; 6(7): 1135 - 1146.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
T. M. Hinton, M. J. Coldwell, G. A. Carpenter, S. J. Morley, and V. M. Pain
Functional Analysis of Individual Binding Activities of the Scaffold Protein eIF4G
J. Biol. Chem., January 19, 2007; 282(3): 1695 - 1708.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
A. Castello, E. Alvarez, and L. Carrasco
Differential Cleavage of eIF4GI and eIF4GII in Mammalian Cells: EFFECTS ON TRANSLATION
J. Biol. Chem., November 3, 2006; 281(44): 33206 - 33216.
[Abstract] [Full Text] [PDF]

Home page
 RNAHome page
S. Miyakawa, A. Oguro, T. Ohtsu, H. Imataka, N. Sonenberg, and Y. Nakamura
RNA aptamers to mammalian initiation factor 4G inhibit cap-dependent translation by blocking the formation of initiation factor complexes
RNA, October 1, 2006; 12(10): 1825 - 1834.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
A. K. LeFebvre, N. L. Korneeva, M. Trutschl, U. Cvek, R. D. Duzan, C. A. Bradley, J. W. B. Hershey, and R. E. Rhoads
Translation Initiation Factor eIF4G-1 Binds to eIF3 through the eIF3e Subunit
J. Biol. Chem., August 11, 2006; 281(32): 22917 - 22932.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. Biol.Home page
A. V. Jivotovskaya, L. Valasek, A. G. Hinnebusch, and K. H. Nielsen
Eukaryotic Translation Initiation Factor 3 (eIF3) and eIF2 Can Promote mRNA Binding to 40S Subunits Independently of eIF4G in Yeast
Mol. Cell. Biol., February 15, 2006; 26(4): 1355 - 1372.
[Abstract] [Full Text] [PDF]

Home page