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

2000; Elsevier BV; Volume: 275; Issue: 52 Linguagem: Inglês

10.1074/jbc.m007525200

ISSN

1083-351X

Autores

Nadia L. Korneeva, Barry J. Lamphear, F. L. Colby Hennigan, Robert E. Rhoads,

Tópico(s)

RNA regulation and disease

Resumo

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 × 104m−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. 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 × 104m−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. methionyl initiator tRNA eukaryotic initiation factor amino acid residue molar binding ratio of eIF4A or eIF3 to eIF4G fragments encephalomyocarditis virus internal ribosomal entry site poly(A)-binding protein polyacrylamide gel electrophoresis response unit(s) surface plasmon resonance 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 (1Merrick W.C. Hershey J.W.B. Hershey J.W.B. Mathews M.B. Sonenberg N. Translational Control. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1996: 31-69Google Scholar). 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 (2Pestova T.V. Lomakin I.B. Lee J.H. Choi S.K. Dever T.E. Hellen C.U.T. Nature. 2000; 403: 332-335Crossref PubMed Scopus (314) Google Scholar). eIF1 and eIF1A act synergistically to mediate assembly of initiation complexes at the initiation codon (3Pestova T.V. Borukhov S.I. Hellen C.U.T. Nature. 1998; 394: 854-859Crossref PubMed Scopus (325) Google Scholar). 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 (4Benne R. Hershey J.W.B. J. Biol. Chem. 1978; 253: 3078-3087Abstract Full Text PDF PubMed Google Scholar, 5Trachsel H. Staehelin T. Biochim. Biophys. Acta. 1979; 565: 305-315Crossref PubMed Scopus (72) Google Scholar, 6Chaudhuri J. Chowdhury D. Maitra U. J. Biol. Chem. 1999; 274: 17975-17980Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Mammalian eIF3 contains 10 non-identical polypeptides termed p170, p116, p110, p66, p48, p47, p44, p40, p36, and p35 (7Hershey J.W.B. Asano K. Naranda T. Vornlocher H.P. Hanachi P. Merrick W.C. Biochimie (Paris). 1996; 78: 903-907Crossref PubMed Scopus (61) Google Scholar, 8Asano K. Kinzy T.G. Merrick W.C. Hershey J.W. J. Biol. Chem. 1997; 272: 1101-1109Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). Five of these polypeptides have identifiable homologs inSaccharomyces cerevisiae (8Asano K. Kinzy T.G. Merrick W.C. Hershey J.W. J. Biol. Chem. 1997; 272: 1101-1109Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 9Phan L. Zhang X. Asano K. Anderson J. Vornlocher H.-P. Greenberg J.R. Qin J. Hinnebusch A.G. Mol. Cell. Biol. 1998; 18: 4935-4946Crossref PubMed Scopus (159) Google Scholar). 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 (10Brown-Luedi M.L. Meyer L.J. Milburn S.C. Yau P.M.P. Corbett S. Hershey J.W.B. Biochemistry. 1982; 21: 4202-4206Crossref PubMed Scopus (52) Google Scholar). At least five subunits of mammalian eIF3, p170, p116 or p110, 2These two eIF3 subunits migrate at the same position under most SDS-PAGE conditions; it is unknown which of them binds RNA. p66, p47, and p44, bind RNA (11Westermann P. Nygard O. Nucleic Acids Res. 1984; 12: 8887-8897Crossref PubMed Scopus (50) Google Scholar, 12Setyono B. Van Steeg H. Voorma H.O. Biochim. Biophys. Acta. 1984; 782: 242-246Crossref PubMed Scopus (14) Google Scholar, 13Westermann P. Sohi M.K. Arnstein H.R.V. FEBS Lett. 1986; 205: 171-174Crossref PubMed Scopus (8) Google Scholar, 14Sizova D.V. Kolupaeva V.G. Pestova T.V. Shatsky I.N. Hellen C.U. J. Virol. 1998; 72: 4775-4782Crossref PubMed Google Scholar, 15Block K.L. Vornlocher H.P. Hershey J.W. J. Biol. Chem. 1998; 273: 31901-31908Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 16Asano K. Vornlocher H.-P. Richter-Cook N.J. Merrick W.C. Hinnebusch A.G. Hershey J.W.B. J. Biol. Chem. 1997; 272: 27042-27052Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, 17Oderman-Macchioli F.E. Tisminetzky S.G. Zotti M. Baralle F.E. Buratti E. Nucleic Acids Res. 2000; 28: 875-885Crossref PubMed Scopus (56) Google Scholar). Of these, p66 and p44 have been shown to bind 18 S ribosomal RNA (13Westermann P. Sohi M.K. Arnstein H.R.V. FEBS Lett. 1986; 205: 171-174Crossref PubMed Scopus (8) Google Scholar, 15Block K.L. Vornlocher H.P. Hershey J.W. J. Biol. Chem. 1998; 273: 31901-31908Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 18Nygard O. Westermann P. Nucleic Acids Res. 1982; 10: 1327-1334Crossref PubMed Scopus (38) Google Scholar). Mammalian eIF3 also cross-links to 40 S ribosomal subunit proteins (19Tolan D.R. Hershey J.W. Traut R.T. Biochimie (Paris). 1983; 65: 427-436Crossref PubMed Scopus (59) Google Scholar). Mammalian eIF3 binds eIF1 via p110 (20Fletcher C.M. Pestova T.V. Hellen C.U. Wagner G. EMBO J. 1999; 18: 2631-2637Crossref PubMed Scopus (109) Google Scholar), eIF4B via p170 (21Methot N. Song M.S. Sonenberg N. Mol. Cell. Biol. 1996; 16: 5328-5334Crossref PubMed Scopus (157) Google Scholar), and eIF5 (22Bandyopadhyay A. Maitra U. Nucleic Acids Res. 1999; 27: 1331-1337Crossref PubMed Scopus (31) Google Scholar). Yeast eIF3 binds both eIF1 and eIF5 via Nip1p (9Phan L. Zhang X. Asano K. Anderson J. Vornlocher H.-P. Greenberg J.R. Qin J. Hinnebusch A.G. Mol. Cell. Biol. 1998; 18: 4935-4946Crossref PubMed Scopus (159) Google Scholar) and eIF4B via p33 (23Vornlocher H.P. Hanachi P. Ribeiro S. Hershey J.W. J. Biol. Chem. 1999; 274: 16802-16812Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Mammalian eIF3 also interacts with the eIF4F complex (which consists of eIF4E, eIF4A, and eIF4G) via eIF4G (24Lamphear B.J. Kirchweger R. Skern T. Rhoads R.E. J. Biol. Chem. 1995; 270: 21975-21983Abstract Full Text Full Text PDF PubMed Scopus (475) Google Scholar). A model whereby eIF3 serves as a bridge between the 40 S ribosomal subunit and the eIF4F·mRNA complex has been postulated (24Lamphear B.J. Kirchweger R. Skern T. Rhoads R.E. J. Biol. Chem. 1995; 270: 21975-21983Abstract Full Text Full Text PDF PubMed Scopus (475) Google Scholar). eIF4G is the central linking protein for all initiation factors known to be involved in mRNA recruitment to the ribosome (25Keiper B.D. Gan W. Rhoads R.E. Int. J. Biochem. Cell Biol. 1999; 31: 37-41Crossref PubMed Scopus (58) Google Scholar). eIF4G binds the cap-binding protein eIF4E (24Lamphear B.J. Kirchweger R. Skern T. Rhoads R.E. J. Biol. Chem. 1995; 270: 21975-21983Abstract Full Text Full Text PDF PubMed Scopus (475) Google Scholar, 26Mader S. Lee H. Pause A. Sonenberg N. Mol. Cell. Biol. 1995; 15: 4990-4997Crossref PubMed Google Scholar, 27Metz A.M. Browning K. J. Biol. Chem. 1996; 271: 31033-31036Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar), PABP (28Imataka H. Gradi A. Sonenberg N. EMBO J. 1998; 17: 7480-7489Crossref PubMed Scopus (475) Google Scholar, 29Tarun S.Z. Sachs A.B. EMBO J. 1996; 15: 7168-7177Crossref PubMed Scopus (585) Google Scholar, 30Le H. Tanguay R.L. Balasta M.L. Wei C.C. Browning K. Metz A.M. Goss D.J. Gallie D.R. J. Biol. Chem. 1997; 272: 16247-16255Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar), eIF4A (24Lamphear B.J. Kirchweger R. Skern T. Rhoads R.E. J. Biol. Chem. 1995; 270: 21975-21983Abstract Full Text Full Text PDF PubMed Scopus (475) Google Scholar, 31Imataka H. Sonenberg N. Mol. Cell. Biol. 1997; 17: 6940-6947Crossref PubMed Scopus (241) Google Scholar, 32Neff C.L. Sachs A.B. Mol. Cell. Biol. 1999; 19: 5557-5564Crossref PubMed Scopus (53) Google Scholar, 33Dominguez D. Altmann M. Benz J. Baumann U. Trachsel H. J. Biol. Chem. 1999; 274: 26720-26726Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar), eIF3 (24Lamphear B.J. Kirchweger R. Skern T. Rhoads R.E. J. Biol. Chem. 1995; 270: 21975-21983Abstract Full Text Full Text PDF PubMed Scopus (475) Google Scholar), the eIF4E-kinase Mnk1 (34Pyronnet S. Imataka H. Gingras A.-C. Fukunaga R. Hunter T. Sonenberg N. EMBO J. 1999; 18: 270-279Crossref PubMed Scopus (543) Google Scholar, 35Waskiewicz A.J. Johnson J.C. Penn B. Mahalingam M. Kimball S.R. Cooper J.A. Mol. Cell. Biol. 1999; 19: 1871-1880Crossref PubMed Scopus (405) Google Scholar), and both mRNA (36Goyer C. Altmann M. Lee H.S. Blanc A. Deshmukh M. Woolford Jr., J.L. Trachsel H. Sonenberg N. Mol. Cell. Biol. 1993; 13: 4860-4874Crossref PubMed Scopus (149) Google Scholar) and the EMCV IRES (37Kolupaeva V.G. Pestova T.V. Hellen C. Shatsky I.N. J. Biol. Chem. 1998; 273: 18599-18604Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 38Pestova T.V. Shatsky I.N. Hellen C.U. Mol. Cell. Biol. 1996; 16: 6870-6878Crossref PubMed Scopus (303) Google Scholar). 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) (24Lamphear B.J. Kirchweger R. Skern T. Rhoads R.E. J. Biol. Chem. 1995; 270: 21975-21983Abstract Full Text Full Text PDF PubMed Scopus (475) Google Scholar). 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 (39Lamphear B.J. Yan R. Yang F. Waters D. Liebig H.-D. Klump H. Kuechler E. Skern T. Rhoads R.E. J. Biol. Chem. 1993; 268: 19200-19203Abstract Full Text PDF PubMed Google Scholar, 24Lamphear B.J. Kirchweger R. Skern T. Rhoads R.E. J. Biol. Chem. 1995; 270: 21975-21983Abstract Full Text Full Text PDF PubMed Scopus (475) Google Scholar). 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 (40De Gregorio E. Preiss T. Hentze M.W. EMBO J. 1999; 18: 4865-4874Crossref PubMed Scopus (137) Google Scholar) and in vitro(38Pestova T.V. Shatsky I.N. Hellen C.U. Mol. Cell. Biol. 1996; 16: 6870-6878Crossref PubMed Scopus (303) Google Scholar), whereas the COOH-terminal domain has been proposed to serve as regulatory domain (41Morino S. Imataka H. Svitkin Y.V. Pestova T.V. Sonenberg N. Mol. Cell. Biol. 2000; 20: 468-477Crossref PubMed Scopus (168) Google Scholar, 42Korneeva, N. L., Lamphear, B. J., Hennigan, F. L. C., Merrick, W. C., and Rhoads, R. E. (2001) J. Biol. Chem. 276, in press.Google Scholar). 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. 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-β-d-thiogalactoside was obtained from Indofine Chemical Co. (Belle Mead, NJ). Preparation of the plasmids pTS4G-(613–1560), pTS4G-(613–1078), pTS4G-(1078–1560), pTS4G-(877–1078), and pTS4G-(975–1078) is described elsewhere (42Korneeva, N. L., Lamphear, B. J., Hennigan, F. L. C., Merrick, W. C., and Rhoads, R. E. (2001) J. Biol. Chem. 276, in press.Google Scholar). Purification and14C labeling of eIF4A by reductive methylation was performed as described previously (43Yoder-Hill J. Pause A. Sonenberg N. Merrick W.C. J. Biol. Chem. 1993; 268: 5566-5573Abstract Full Text PDF PubMed Google Scholar). eIF3, eIF4A, and eIF4F were purified from the ribosomal high salt wash of rabbit reticulocyte lysate by m7GTP-Sepharose and Mono Q chromatography (44Lamphear B.J. Panniers R. J. Biol. Chem. 1990; 265: 5333-5336Abstract Full Text PDF PubMed Google Scholar). 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 β-mercaptoethanol, 0.1% (v/v) Tween 20, and 2 mm EDTA, pH 7.5) plus 5% (v/v) glycerol. 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 (42Korneeva, N. L., Lamphear, B. J., Hennigan, F. L. C., Merrick, W. C., and Rhoads, R. E. (2001) J. Biol. Chem. 276, in press.Google Scholar) 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 (42Korneeva, N. L., Lamphear, B. J., Hennigan, F. L. C., Merrick, W. C., and Rhoads, R. E. (2001) J. Biol. Chem. 276, in press.Google Scholar) 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 (45Liebig 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. Skern T. Biochemistry. 1993; 32: 7581-7588Crossref PubMed Scopus (167) Google Scholar) 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 mmEDTA, 0.05% (v/v) Tween 20, and 0.5 mmβ-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. RNA corresponding to the EMCV IRES was transcribed in vitro using the Promega Riboprobe system. Briefly, transcription reactions (20 μl) containingBglII-linearized pCite4Gwt (1 μg) (46Lamphear B.J. Rhoads R.E. Biochemistry. 1996; 35: 15726-15733Crossref PubMed Scopus (30) Google Scholar), 0.5 mm GTP, 0.5 mm CTP, 0.5 mm ATP, 20 μm UTP, 30 μCi of [α-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. 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 of32P-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. 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 (47Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (212382) Google Scholar), with detection by Coomassie Blue staining. 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 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[eIF3] f/(Kd+[eIF3] f)Equation 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 (48Scatchard G. Ann. N. Y. Acad. Sci. 1949; 51: 600-672Crossref Scopus (18122) Google Scholar). 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[eIF4A] f/(Kd+[eIF4A] f)Equation 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 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 mmsodium 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 (49Stenberg E. Persson B. Roos H. Urbaniczky C. J. Colloid Interface Sci. 1991; 143: 513-526Crossref Scopus (1014) Google Scholar). 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 β-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 ⇔ AB) as described elsewhere (42Korneeva, N. L., Lamphear, B. J., Hennigan, F. L. C., Merrick, W. C., and Rhoads, R. E. (2001) J. Biol. Chem. 276, in press.Google Scholar). Values for the statistical closeness of fit, χ2, were always below 10, indicating that the simple 1:1 model of interaction correctly described the experimental data. An earlier study indicated that eIF3 binds to the region of human eIF4G from aa 635 to 1041, which constitutes the central domain (24Lamphear B.J. Kirchweger R. Skern T. Rhoads R.E. J. Biol. Chem. 1995; 270: 21975-21983Abstract Full Text Full Text PDF PubMed Scopus (475) Google Scholar). This was confirmed in subsequent studies with eIF4G fragments representing aa 613–1090 (31Imataka H. Sonenberg N. Mol. Cell. Biol. 1997; 17: 6940-6947Crossref PubMed Scopus (241) Google Scholar) and aa 672–1065 (41Morino S. Imataka H. Svitkin Y.V. Pestova T.V. Sonenberg N. Mol. Cell. Biol. 2000; 20: 468-477Crossref PubMed Scopus (168) Google Scholar). To characterize the eIF3-binding site further, S-eIF4G fragments of decreasing size (Fig. 1 A) 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. 1 B, 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).Figure 1A, 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 proindicates the site of entero- and rhinoviral 2A protease cleavage (39Lamphear B.J. Yan R. Yang F. Waters D. Liebig H.-D. Klump H. Kuechler E. Skern T. Rhoads R.E. J. Biol. Chem. 1993; 268: 19200-19203Abstract Full Text PDF PubMed Google Scholar). 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.View Large Image Figure ViewerDownload (PPT) 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. 1 A.) 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 eIF4E 3Detection 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. (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. The central domain of eIF4G binds both eIF3 (24Lamphear B.J. Kirchweger R. Skern T. Rhoads R.E. J. Biol. Chem. 1995; 270: 21975-21983Abstract Full Text Full Text PDF PubMed Scopus (475) Google Scholar) and the EMCV IRES (38Pestova T.V. Shatsky I.N. Hellen C.U. Mol. Cell. Biol. 1996; 16: 6870-6878Crossref PubMed Scopus (303) Google Scholar, 37Kolupaeva V.G. Pestova T.V. Hellen C. Shatsky I.N. J. Biol. Chem. 1998; 273: 18599-18604Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). 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. 3 B, 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 si

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