Abstract: The original purification of the heterotrimeric eIF4F was published over 30 years ago (Grifo, J. A., Tahara, S. M., Morgan, M. A., Shatkin, A. J., and Merrick, W. C. (1983) J. Biol. Chem. 258, 5804–5810). Since that time, numerous studies have been performed with the three proteins specifically required for the translation initiation of natural mRNAs, eIF4A, eIF4B, and eIF4F. These have involved enzymatic and structural studies of the proteins and a number of site-directed mutagenesis studies. The regulation of translation exhibited through the mammalian target of rapamycin (mTOR) pathway is predominately seen as the phosphorylation of 4E-BP, an inhibitor of protein synthesis that functions by binding to the cap binding subunit of eIF4F (eIF4E). A hypothesis that requires the disassembly of eIF4F during translation initiation to yield free subunits (eIF4A, eIF4E, and eIF4G) is presented. The original purification of the heterotrimeric eIF4F was published over 30 years ago (Grifo, J. A., Tahara, S. M., Morgan, M. A., Shatkin, A. J., and Merrick, W. C. (1983) J. Biol. Chem. 258, 5804–5810). Since that time, numerous studies have been performed with the three proteins specifically required for the translation initiation of natural mRNAs, eIF4A, eIF4B, and eIF4F. These have involved enzymatic and structural studies of the proteins and a number of site-directed mutagenesis studies. The regulation of translation exhibited through the mammalian target of rapamycin (mTOR) pathway is predominately seen as the phosphorylation of 4E-BP, an inhibitor of protein synthesis that functions by binding to the cap binding subunit of eIF4F (eIF4E). A hypothesis that requires the disassembly of eIF4F during translation initiation to yield free subunits (eIF4A, eIF4E, and eIF4G) is presented. The initial findings in the study of natural mRNA translation reflected the newly discovered m7G cap at the 5′ end of eukaryotic mRNAs (2Both G.W. Furuichi Y. Muthukrishnan S. Shatkin A.J. Ribosome binding to reovirus mRNA in protein synthesis requires 5′ terminal 7-methylguanosine.Cell. 1975; 6: 185-195Abstract Full Text PDF PubMed Scopus (140) Google Scholar). mRNAs lacking this structure were translated with less efficiency than mRNAs that contained this structure (3Zan-Kowalczewska M. Bretner M. Sierakowska H. Szczesna E. Filipowicz W. Shatkin A.J. Removal of the 5′-terminal m7G from eukaryotic mRNAs by potato nucleotide pyrophosphatase and its effect on translation.Nucleic Acids Res. 1977; 4: 3065-3081Crossref PubMed Scopus (39) Google Scholar). This unique structure allowed for specific assays or purifications, many established in the laboratory of Dr. Aaron Shatkin with assists from his colleagues. Two of note were the use of m7G-Sepharose for affinity purification (4Sonenberg N. Rupprecht K.M. Hecht S.M. Shatkin A.J. Eukaryotic mRNA cap binding protein: purification by affinity chromatography on Sepharose-coupled m7GDP.Proc. Natl. Acad. Sci. U.S.A. 1979; 76: 4345-4349Crossref PubMed Scopus (164) Google Scholar, 5Webb N.R. Chari R.V. DePillis G. Kozarich J.W. Rhoads R.E. Purification of the messenger RNA cap-binding protein using a new affinity medium.Biochemistry. 1984; 23: 177-181Crossref PubMed Scopus (64) Google Scholar) and the crosslinking of periodate-oxidized mRNAs to proteins (6Sonenberg N. Shatkin A.J. Reovirus mRNA can be covalently crosslinked via the 5′ cap to proteins in initiation complexes.Proc. Natl. Acad. Sci. U.S.A. 1977; 74: 4288-4292Crossref PubMed Scopus (44) Google Scholar). The initial application of these methodologies identified two different molecular weight species (about 25,000 and at least 200,000), although the high molecular weight protein contained the small molecular weight component, now known as eIF4E (7Tahara S.M. Morgan M.A. Shatkin A.J. Two forms of purified m7G-cap binding protein with different effects on capped mRNA translation in extracts of uninfected and poliovirus-infected HeLa cells.J. Biol. Chem. 1981; 256: 7691-7694Abstract Full Text PDF PubMed Google Scholar). Given the size of several other known translation factors, the question was whether these contained the small molecular weight subunit (notably eIF3 and eIF4B) (8Padilla M. Canaani D. Groner Y. Weinstein J.A. Bar-Joseph M. Merrick W. Shafritz D.A. Initiation factor eIF-4B (IF-M3)-dependent recognition and translation of capped versus uncapped eukaryotic mRNAs.J. Biol. Chem. 1978; 253: 5939-5945Abstract Full Text PDF PubMed Google Scholar, 9Hansen J. Etchison D. 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Ultimate purification of eIF4F indicated that neither of these were correct but that eIF4F would form stable complexes with either, thus being consistent with the eIF4E component tracking with them. At the same time, it was recognized that the 46,000 molecular weight subunit of eIF4F was eIF4A. By physical analysis, eIF4F was a heterotrimeric complex of eIF4A, eIF4E, and eIF4G (1Grifo J.A. Tahara S.M. Morgan M.A. Shatkin A.J. Merrick W.C. New initiation factor activity required for globin mRNA translation.J. Biol. Chem. 1983; 258: 5804-5810Abstract Full Text PDF PubMed Google Scholar). The next studies were to attempt to identify the functions of the various proteins required specifically for natural mRNA translation (eIF4A, eIF4B, and eIF4F). The characteristics of these three proteins were very different. In the absence of ATP, binding to RNA could only be well demonstrated with eIF4F (Table 1). eIF4A and eIF4F could hydrolyze ATP in the presence of single-stranded RNA, and eIF4B would enhance both of these activities (12Abramson R.D. Dever T.E. Lawson T.G. Ray B.K. Thach R.E. Merrick W.C. The ATP-dependent interaction of eukaryotic initiation factors with mRNA.J. Biol. Chem. 1987; 262: 3826-3832Abstract Full Text PDF PubMed Google Scholar). In terms of mechanism, the coupling of the binding of ATP and RNA was realized in recognizing that eIF4A or eIF4F had the ability to unwind duplex RNA. As noted in Table 1, the “strength” of the helicase activity was greater with eIF4F (14Rogers Jr., G.W. Richter N.J. Lima W.F. Merrick W.C. Modulation of the helicase activity of eIF4A by eIF4B, eIF4H, and eIF4F.J. Biol. Chem. 2001; 276: 30914-30922Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar).TABLE 1Relative activities of translation initiation factorsInitiation factorGlobin mRNA bindingaRadioactively labeled globin mRNA retention on nitrocellulose filters (cpm) (12).RNA-dependent ATPasebHydrolysis of [γ-32P]ATP in response to added RNA (Kact in μm, Vmax in fmol of PO4 released per s per μg of either eIF4A or eIF4F) (13).Helicase activitycUnwinding of duplex RNA with a 33-nucleotide single-stranded region or 29-nucleotide single-stranded region; duplexes were 11 and 15 bp, respectively (initial rate of unwinding) (14).−ATP+ATPKactVmaxΔG = −17.9ΔG = −24.7eIF4A130180015,0001103.00.6eIF4B230350–d— = not determined.–d— = not determined.–d— = not determined.–d— = not determined.eIF4F3610399030207.31.3eIF4A, eIF4B15051706013510.33.6eIF4F, eIF4B3820426030309.63.6a Radioactively labeled globin mRNA retention on nitrocellulose filters (cpm) (12Abramson R.D. Dever T.E. Lawson T.G. Ray B.K. Thach R.E. Merrick W.C. The ATP-dependent interaction of eukaryotic initiation factors with mRNA.J. Biol. Chem. 1987; 262: 3826-3832Abstract Full Text PDF PubMed Google Scholar).b Hydrolysis of [γ-32P]ATP in response to added RNA (Kact in μm, Vmax in fmol of PO4 released per s per μg of either eIF4A or eIF4F) (13Abramson R.D. Dever T.E. Merrick W.C. Biochemical evidence supporting a mechanism for cap-independent and internal initiation of eukaryotic mRNA.J. Biol. Chem. 1988; 263: 6016-6019Abstract Full Text PDF PubMed Google Scholar).c Unwinding of duplex RNA with a 33-nucleotide single-stranded region or 29-nucleotide single-stranded region; duplexes were 11 and 15 bp, respectively (initial rate of unwinding) (14Rogers Jr., G.W. Richter N.J. Lima W.F. Merrick W.C. Modulation of the helicase activity of eIF4A by eIF4B, eIF4H, and eIF4F.J. Biol. Chem. 2001; 276: 30914-30922Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar).d — = not determined. Open table in a new tab As the ability to determine amino acid sequence from RNA sequence advanced, it was found that there were numerous conserved amino acid motifs in eIF4A that could be found in other proteins, and this led to the establishment of the DEAD-box proteins (15Linder P. Lasko P.F. Ashburner M. Leroy P. Nielsen P.J. Nishi K. Schnier J. Slonimski P.P. Birth of the D-E-A-D box.Nature. 1989; 337: 121-122Crossref PubMed Scopus (629) Google Scholar). The DEAD-box proteins became the first group of well characterized RNA helicases. This information was soon put into a variety of model initiation pathways in which the primary feature of the eIF4 proteins was their utilization for the unwinding of possible secondary structure to form a single-stranded RNA that could be bound to the 40S subunit (as the 43S preinitiation complex containing eIF1, eIF1A, eIF3, and the ternary complex eIF2·GTP·Met-tRNAi). A later finding that RNA helicases can displace protein from an RNA·protein complex adds a second element to the activation process as mRNAs exit the nucleus as mRNPs 2The abbreviations used are: mRNPmessenger ribonucleoproteinmTORmammalian target of rapamycinPABPpoly(A)-binding proteinADPNP5′-adenylyl-β,γ-imidodiphosphate4E-BPeIF4E-binding protein. (16Jankowsky E. Gross C.H. Shuman S. Pyle A.M. Active disruption of an RNA-protein interaction by a DExH/D RNA helicase.Science. 2001; 291: 121-125Crossref PubMed Scopus (253) Google Scholar). messenger ribonucleoprotein mammalian target of rapamycin poly(A)-binding protein 5′-adenylyl-β,γ-imidodiphosphate eIF4E-binding protein. In the mid-90s, as efforts were continuing to define the biochemistry of the eIF4 family proteins, a very unique discovery was made. One of the major proteins to be phosphorylated in cells in response to insulin treatment was called PHAS-I (phosphorylated heat- and acid-stable protein regulated by insulin) (17Hu C. Pang S. Kong X. Velleca M. Lawrence Jr, J.C. Molecular cloning and tissue distribution of PHAS-I, an intracellular target for insulin and growth factors.Proc. Natl. Acad. Sci. U.S.A. 1994; 91: 3730-3734Crossref PubMed Scopus (125) Google Scholar). By a separate analytic procedure, this protein was also identified as a protein that bound to eIF4E and would later be discovered to be a major target of the mTOR pathway (mTORC1) (18Pause A. Belsham G.J. Gingras A.C. Donzé O. Lin T.A. Lawrence Jr., J.C. Sonenberg N. Insulin-dependent stimulation of protein synthesis by phosphorylation of a regulator of 5′-cap function.Nature. 1994; 371: 762-767Crossref PubMed Scopus (1055) Google Scholar). The phosphorylation of this protein (4E-BP) led to its inactivation (inability to bind to eIF4E). Because the binding of eIF4E by either eIF4G or 4E-BP is to overlapping sites on eIF4E, only one of the two can be bound to eIF4E at any point in time (19Lukhele S. Bah A. Lin H. Sonenberg N. Forman-Kay J.D. Interaction of the eukaryotic initiation factor 4E with 4E-BP2 at a dynamic bipartite interface.Structure. 2013; 21: 2186-2196Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 20Peter D. Igreja C. Weber R. Wohlbold L. Weiler C. Ebertsch L. Weichenrieder O. Izaurralde E. Molecular architecture of 4E-BP translational inhibitors bound to eIF4E.Mol. Cell. 2015; 57: 1074-1087Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). This finding added a second arm to the global regulation of eukaryotic protein synthesis. The first was the regulation of available initiator tRNA as the ternary complex eIF2·GTP·Met-tRNAi. The second was the restriction in the level of eIF4F activity due to the lowered availability of eIF4E. Consistent with the general pathway of 80S complex formation (Fig. 1) was that the binding of the ternary complex preceded the binding of mRNA. As a consequence, translation of most mRNAs was reduced equally when ternary complexes became limiting. In contrast, reduction of eIF4F activity forced mRNAs to compete for limiting eIF4F, and this resulted in “good” mRNAs being preferentially translated, whereas “poor” mRNAs were not. This was consistent with the mathematical modeling, initially by Lodish and colleagues (21Temple G. Lodish H.F. Competition between α and β globin messenger RNA.Biochem. Biophys. Res. Comm. 1975; 63: 971-979Crossref PubMed Scopus (15) Google Scholar, 22Bergmann J.E. Lodish H.F. A kinetic model of protein synthesis: application to hemoglobin synthesis and translational control.J. Biol. Chem. 1979; 254: 11927-11937Abstract Full Text PDF PubMed Google Scholar) and then refined by Godefroy-Colburn and Thach (23Godefroy-Colburn T. Thach R.E. The role of mRNA competition in regulating translation. IV Kinetic model.J. Biol. Chem. 1981; 256: 11762-11773Abstract Full Text PDF PubMed Google Scholar). Although a generalization, it would appear that the ability of an mRNA to compete for the translational apparatus was mostly determined by the availability of its m7G cap (24Godefroy-Colburn T. Ravelonandro M. Pinck L. Cap accessibility correlates with the initiation efficiency of alfalfa mosaic virus RNAs.Eur. J. Biochem. 1985; 147: 549-552Crossref PubMed Scopus (24) Google Scholar). A second protein that can influence the formation of eIF4F is the protein PDCD4, a tumor suppressor. This protein binds to eIF4A and can thereby limit the amount of eIF4A available to form eIF4F (25Yang H.S. Jansen A.P. Komar A.A. Zheng X. Merrick W.C. Costes S. Lockett S.J. Sonenberg N. Colburn N.H. The transformation suppressor Pdcd4 is a novel eukaryotic translation initiation factor 4A binding protein that inhibits translation.Mol. Cell. Biol. 2003; 23: 26-37Crossref PubMed Scopus (408) Google Scholar). PDCD4 is regulated through phosphorylation by the mTOR pathway (which inhibits its binding to eIF4A) (26Suzuki C. Garces R.G. Edmonds K.A. Hiller S. Hyberts S.G. Marintchev A. Wagner G. PDCD4 inhibits translation initiation by binding to eIF4A using both its MA3 domains.Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 3274-3279Crossref PubMed Scopus (116) Google Scholar, 27Chang J.H. Cho Y.H. Sohn S.Y. Choi J.M. Kim A. Kim Y.C. Jang S.K. Cho Y. Crystal structure of the eIF4A-PDCD4 complex.Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 3148-3153Crossref PubMed Scopus (74) Google Scholar, 28Dennis M.D. Jefferson L.S. Kimball S.R. Role of p70S6K1-mediated phosphorylation of eIF4B and PDCD4 proteins in the regulation of protein synthesis.J. Biol. Chem. 2012; 287: 42890-42899Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). Based on the observation that mRNAs with structured 5′ ends require additional eIF4A for translation, the lack of PDCD4 phosphorylation could lead to reduced translation of these mRNAs, either as free eIF4A or as eIF4A to reconstitute eIF4F. In sum, mTOR coordinately regulates eIF4F formation by controlling the level of phosphorylation of both 4E-BP and PDCD4. With the recognition that eIF4A was a subunit of eIF4F, much of the subsequent biochemistry focused on the difference between these two proteins and the differential affect of eIF4B on these proteins (it is noted that the relatively late discovery of eIF4H has resulted in more minimal studies in this comparison) (29Richter N.J. Rogers Jr., G.W. Hensold J.O. Merrick W.C. Further biochemical and kinetic characterization of human eukaryotic initiation factor 4H.J. Biol. Chem. 1999; 274: 35415-35424Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). The standard assays initially used were: 1) RNA binding, an assay that monitors the retention of an RNA on nitrocellulose filters indicative of a protein·nucleic acid complex; 2) RNA-dependent ATPase, an assay that measures the hydrolysis of ATP in a reaction requiring the presence of RNA; and 3) duplex RNA unwinding, an assay that measures the ATP-dependent strand separation of an RNA duplex to yield two single strands. For a number of RNA helicases, there is a dependence on a single-stranded region being part of the duplex. Data from these assays are shown in Table 1. Consistent with their behavior on phosphocellulose, in the absence of ATP, eIF4A failed to bind mRNA, whereas eIF4F bound significant levels of mRNA (30Merrick W.C. Purification of protein synthesis initiation factors from rabbit reticulocytes.Methods Enzymol. 1979; 60: 101-108Crossref PubMed Scopus (67) Google Scholar). However, the presence of ATP greatly enhanced the binding of mRNA by eIF4A (with or without eIF4B), whereas it only offered a slight stimulation with eIF4F. The kinetic data for the RNA-dependent ATPase assay indicate that the primary difference between eIF4A and eIF4F is the apparent affinity of the proteins for RNA, although the presence of eIF4B renders the ability of eIF4A to catalyze hydrolysis nearly equivalent to that seen with eIF4F (13Abramson R.D. Dever T.E. Merrick W.C. Biochemical evidence supporting a mechanism for cap-independent and internal initiation of eukaryotic mRNA.J. Biol. Chem. 1988; 263: 6016-6019Abstract Full Text PDF PubMed Google Scholar). Similar trends are evident when the melting of RNA duplexes is considered; however, the -fold difference in the initial rates of unwinding is less dramatic with a 2–3-fold difference between eIF4A and eIF4F (± eIF4B) with a shorter duplex, but a greater difference with a more stable duplex (up to 6-fold) (14Rogers Jr., G.W. Richter N.J. Lima W.F. Merrick W.C. Modulation of the helicase activity of eIF4A by eIF4B, eIF4H, and eIF4F.J. Biol. Chem. 2001; 276: 30914-30922Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar). A relatively early observation was that Saccharomyces cerevisiae eIF4F is different from the human protein and that some of these differences play out with eIF4A as well. Perhaps the most challenging difference is that a three-subunit eIF4F has not been purified from yeast, but rather only the two-subunit eIF4E·eIF4G complex has been isolated (31Lanker S. Müller P.P. Altmann M. Goyer C. Sonenberg N. Trachsel H. Interactions of the eIF4F subunits in the yeast Saccharomyces cerevisiae.J. Biol. Chem. 1992; 267: 21167-21171Abstract Full Text PDF PubMed Google Scholar). Secondly, eIF4B is a monomer in yeast but a dimer in the mammalian system, which may have profound influences on the biochemical behavior of either eIF4A or eIF4F (32Altmann M. Wittmer B. Méthot N. Sonenberg N. Trachsel H. The Saccharomyces cerevisiae translation initiation factor Tif3 and its mammalian homologue, eIF4B, have RNA annealing activity.EMBO J. 1995; 14: 3820-3827Crossref PubMed Scopus (85) Google Scholar, 33Grifo J.A. Eukaryotic initiation factors which recognize and bind mRNA. Case Western Reserve University, Cleveland, OH1982Google Scholar, 34Méthot N. Song M.S. Sonenberg N. A region rich in aspartic acid, arginine, tyrosine and glycine (DRYG) mediates eukaryotic initiation factor 4B (eIF4B) self-association and interaction with eIF3.Mol. Cell. Biol. 1996; 16: 5328-5334Crossref PubMed Scopus (156) Google Scholar). In this light, eIF4B enhances the RNA-dependent ATPase activity of eIF4A in the mammalian system by reducing the Kact 1000-fold (13Abramson R.D. Dever T.E. Merrick W.C. Biochemical evidence supporting a mechanism for cap-independent and internal initiation of eukaryotic mRNA.J. Biol. Chem. 1988; 263: 6016-6019Abstract Full Text PDF PubMed Google Scholar). In contrast, there is no stimulation observed in the yeast system. Thus, given these differences, the remaining discussion will focus on the mammalian eIF4F (and eIF4A and eIF4B), although it is anticipated that similar activities will be visualized for the yeast system as well. From numerous studies, interactive domains of eIF4G have been determined, and a graphic representation of these domains is seen in Fig. 2A. Because of the many interactions, eIF4G is able to coordinate functions related to m7G cap binding (eIF4E), RNA helicase unwinding (eIF4A), binding to the 40S subunit (eIF3), and coordinating initiation using freshly terminated ribosomes via the interaction with the poly(A)-binding protein (PABP) as the “circular” mRNA. At the level of crystallographic studies, individual structures for eIF4A, eIF4E, and the HEAT domains of eIF4G have been determined (35Caruthers J.M. Johnson E.R. McKay D.B. Crystal structure of yeast initiation factor 4A, a DEAD-box RNA helicase.Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 13080-13085Crossref PubMed Scopus (244) Google Scholar, 36Meng H. Li C. Wang Y. Chen G. Molecular dynamics simulation of the allosteric regulation of eIF4A protein from the open to closed state, induced by ATP and RNA substrates.PLoS One. 2014; 9e86104Crossref PubMed Scopus (9) Google Scholar, 37Marcotrigiano J. Lomakin I.B. Sonenberg N. Pestova T.V. Hellen C.U.T. Burley S.K. A conserved HEAT domain with eIF4G directs assembly of the translation initiation machinery.Mol. Cell. 2001; 7: 193-203Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 38Oberer M. Marintchev A. Wagner G. Structural basis for the enhancement of the eIF4A helicase activity by eIF4G.Genes Dev. 2005; 19: 2212-2223Crossref PubMed Scopus (120) Google Scholar, 39Bellsolell L. Cho-Park P.F. Poulin F. Sonenberg N. Burley S.K. Two structurally atypical HEAT domains in the C-terminal portion of human eIF4G support binding to eIF4A and Mnk1.Structure. 2006; 14: 913-923Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 40Schütz P. Bumann M. Oberholzer A.E. Bieniossek C. Trachsel H. Altmann M. Baumann U. Crystal structure of the yeast eIF4A-eIF4G complex: an RNA-helicase controlled by protein-protein interactions.Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 9564-9569Crossref PubMed Scopus (174) Google Scholar, 41Marcotrigiano J. Gingras A.C. Sonenberg N. Burley S.K. 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Structure of the eukaryotic translation initiation factor eIF4E in complex with 4EGI-1 reveals an allosteric mechanism for dissociating eIF4G.Proc. Natl. Acad. Sci. U.S.A. 2014; 111: E3187-E3195Crossref PubMed Scopus (57) Google Scholar, 45Feoktistova K. Tuvshintogs E. Do A. Fraser C.S. Human eIF4E promotes mRNA restructuring by stimulating eIF4A helicase activity.Proc. Natl. Acad. Sci. U.S.A. 2013; 110: 13339-13344Crossref PubMed Scopus (111) Google Scholar). The only catalytically active co-crystal structure is for eIF4A complexed with the middle domain of eIF4G (amino acids 572–853), and in this structure, both the N-terminal and the C-terminal domains make contact with the eIF4G HEAT1 domain (Fig. 2B) (37Marcotrigiano J. Lomakin I.B. Sonenberg N. Pestova T.V. Hellen C.U.T. Burley S.K. A conserved HEAT domain with eIF4G directs assembly of the translation initiation machinery.Mol. Cell. 2001; 7: 193-203Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 38Oberer M. Marintchev A. Wagner G. Structural basis for the enhancement of the eIF4A helicase activity by eIF4G.Genes Dev. 2005; 19: 2212-2223Crossref PubMed Scopus (120) Google Scholar, 39Bellsolell L. Cho-Park P.F. Poulin F. Sonenberg N. Burley S.K. Two structurally atypical HEAT domains in the C-terminal portion of human eIF4G support binding to eIF4A and Mnk1.Structure. 2006; 14: 913-923Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 40Schütz P. Bumann M. Oberholzer A.E. Bieniossek C. Trachsel H. Altmann M. Baumann U. Crystal structure of the yeast eIF4A-eIF4G complex: an RNA-helicase controlled by protein-protein interactions.Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 9564-9569Crossref PubMed Scopus (174) Google Scholar). This interaction would appear to restrict the flexibility of the eIF4A molecule around the 11-amino acid flexible linker that joins the two domains. Of particular interest is the effect that eIF4G has on orienting the subdomains of eIF4A into a more active (open) confirmation in contrast to the variety of confirmations theoretically possible with free eIF4A (38Oberer M. Marintchev A. Wagner G. Structural basis for the enhancement of the eIF4A helicase activity by eIF4G.Genes Dev. 2005; 19: 2212-2223Crossref PubMed Scopus (120) Google Scholar, 40Schütz P. Bumann M. Oberholzer A.E. Bieniossek C. Trachsel H. Altmann M. Baumann U. Crystal structure of the yeast eIF4A-eIF4G complex: an RNA-helicase controlled by protein-protein interactions.Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 9564-9569Crossref PubMed Scopus (174) Google Scholar). At present, the mechanism for the observed activation of eIF4A activity by eIF4B is unknown, but it would not be surprising if it were similar. This activation appears to reflect primarily differences in binding nucleic acid as the apparent Km for ATP is relatively unaffected (13Abramson R.D. Dever T.E. Merrick W.C. Biochemical evidence supporting a mechanism for cap-independent and internal initiation of eukaryotic mRNA.J. Biol. Chem. 1988; 263: 6016-6019Abstract Full Text PDF PubMed Google Scholar). An interesting side note comes from the structural analysis of eIF4E complexed with the inhibitor peptide, 4EGI-1 (42Matsuo H. Li H. McGuire A.M. Fletcher C.M. Gingras A.C. Sonenberg N. Wagner G. Structure of translation factor eIF4E bound to m7GDP and interaction with 4E-binding protein.Nat. Struct. Biol. 1997; 4: 717-724Crossref PubMed Scopus (327) Google Scholar, 43Brown C.J. Verma C.S. Walkinshaw M.D. Lane D.P. Crystallization of eIF4E complexed with eIF4GI peptide and glycerol reveals distinct structural differences around the cap-binding site.Cell Cycle. 2009; 8: 1905-1911Crossref PubMed Scopus (30) Google Scholar). In contrast to the 4E-BPs, the inhibitor peptide binds to an allosteric site on eIF4E, leading to the displacement of eIF4G from eIF4E (43Brown C.J. Verma C.S. Walkinshaw M.D. Lane D.P. Crystallization of eIF4E complexed with eIF4GI peptide and glycerol reveals distinct structural differences around the cap-binding site.Cell Cycle. 2009; 8: 1905-1911Crossref PubMed Scopus (30) Google Scholar). Unfortunately, further crystal structure analysis has been limited by either the low level of protein in normal cells or the inability to readily express human full-length eIF4G (although see Feoktistova et al. (45Feoktistova K. Tuvshintogs E. Do A. Fraser C.S. Human eIF4E promotes mRNA restructuring by stimulating eIF4A helicase activity.Proc. Natl. Acad. Sci. U.S.A. 2013; 110: 13339-13344Crossref PubMed Scopus (111) Google Scholar)). This is accompanied by the concern that the post-translational modifications known to occur on both eIF4E and eIF4G may be important for function, and the degree of modification obtained with expression in either Escherichia coli or insect cells may be limiting. Also, as noted for eIF2, it is possible that a cellular protein might be required for the correct assembly of the complete complex (46Perzlmaier A.F. Richter F. Seufert W. Translation initiation requires cell division cycle 123 (Cdc123) to facilitate biogenesis of the eukaryotic initiation factor 2 (eIF2).J. Biol. Chem. 2013; 288: 21537-21546Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). Thus, it will be important to compare both biochemically and physically native protein purified from actively translating systems (i.e. HeLa or reticulocytes) with recombinant proteins. The availability of molecular biological techniques and protein expression has allowed for the preparation of numerous translation initiation factors, either as subunits or as individual proteins. For the most part, these expressed proteins have demonstrated activity either as individual components or as added to a reconstitution assay. The most useful of these are when the expressed protein can be independently assessed for activity, as is the case for eIF4A. With respect to the “mRNA-specific initiation factors,” whereas eIF4E can be assessed for binding to m7GTP-Sepharose and eIF4B and eIF4G can be assessed for binding to nucleic acid, for most of the “functional assays” (RNA-dependent ATPase, RNA duplex unwinding, toe printing, protein synthesis), it is the effect th