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

Abstract

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 ϳ42 nM was derived from an association rate constant of 3.9 ؋ 10 4 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.The initiation of translation in eukaryotes requires multiple initiation factors that stimulate the binding of mRNA and Met-tRNA i 1 to the 40 S ribosomal subunit to form the 48 S preinitiation complex (1). The binding of Met-tRNA i 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 eIF2⅐GDP 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 eIF2⅐GTP⅐Met-tRNA i 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 ϳ600 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 als...

Mass Spectrometric Analysis of the N Terminus of Translational Initiation Factor eIF4G-1 Reveals Novel Isoforms

“…With Arg-C we detected the N-terminal peptide, Peptide r1, and its mass indicated that it was modified in two ways: the N-terminal Met was removed and the resulting Asn was N ␣ -acetylated. Asp-N produced Peptide d1, in agreement with the assignment of eIF4G-1f to Band 1, but it was too large (19,371 Da) to allow determination of the exact N-terminal structure. Thus, all of the data from Band 1 are consistent with its identity as eIF4G-1f.…”

“…Less expected is the presence of the p110 and p36 subunits of eIF3. It is known that 11-subunit eIF3 has a high affinity for eIF4G, but the binding site has been localized in cp C , not cp N (14,19). The association of eIF4G with the p110 and p36 subunits may mean that there are additional points of attachment in cp N .…”

“…eIF4G appears to serve as a nucleation site for co-localization of an unusually large number of proteins involved in mRNA metabolism. Combining data from yeast, plant, and mammalian eIF4Gs, these include (in approximate order of binding sites on eIF4G from N to C termini): the influenza protein NS1 (9), the cytoplasmic poly(A)-binding protein PABP (10,11), the rotavirus protein NSP3 (12), the decapping protein Dcp1 (13), the cytoplasmic cap-binding protein eIF4E (14 -16), the nuclear cap-binding protein CBP80 (17,18), the initiation factor eIF3 (14,19), the RNA helicase eIF4A at two distinct sites (14,20,21), RNA itself (22)(23)(24), the heat shock proteins hsp27 (25) and hsp70 (26), and the eIF4E kinase Mnk1 (27)(28)(29). In some cases it has been shown that eIF4G-1 not only binds proteins but also affects their activities or binding of other proteins (19,21,30).…”

Conducting the initiation of protein synthesis: the role of eIF4G

“…Finally, the exact position of the carboxy-terminus eIF4A binding site has been mapped between aa 1201-1411 by Morino et al (2000) while the upstream binding site is defined as amino acids 672-970 by Morino et al (2000), 672-876 by Korneeva et al (2000Korneeva et al ( , 2001) and 722-949 by Lomakin et al (2000) (see Fig. 2).…”

Plant Cap-binding Complexes Eukaryotic Initiation Factors eIF4F and eIFISO4F

“…Mammalian eIF4G consists of several domains for interaction with these factors. The eIF4E and poly(A) binding protein binding sites are located in the N-terminal region, whereas the C-terminal region contains three HEAT domains, MIF4G (eIF4A and eIF3), MA3 (eIF4A), and W2 (MNK) (1,(13)(14)(15)(16)(17)(18). However, yeast eIF4G has only retained the MIF4G domain and lost the MA3 and W2 HEAT domains.…”