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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...

Eukaryotic translation initiation factor 4G-1 (eIF4G) plays a critical role in the recruitment of mRNA to the 43 S preinitiation complex. eIF4G has two binding sites for the RNA helicase eIF4A, one in the central domain and one in the COOH-terminal domain. Recombinant eIF4G fragments that contained each of these sites separately bound eIF4A with a 1:1 stoichiometry, but fragments containing both sites bound eIF4A with a 1:2 stoichiometry. eIF3 did not interfere with eIF4A binding to the central site. Interestingly, at the same concentration of free eIF4A, more eIF4A was bound to an eIF4G fragment containing both eIF4A sites than the sum of binding to fragments containing the single sites, indicating cooperative binding. Binding of eIF4A to an immobilized fragment of eIF4G containing the COOH-terminal site was competed by a soluble eIF4G fragment containing the central site, indicating that a single eIF4A molecule cannot bind simultaneously to both sites. The association rate constant, dissociation rate constant, and dissociation equilibrium constant for each site were determined by surface plasmon resonance and found to be, respectively, 1. The initiation of translation of most eukaryotic mRNAs involves the sequential recruitment of Met-tRNA i , mRNA, and the 60 S ribosomal subunit to the 40 S ribosomal subunit, catalyzed by the various groups of initiation factors (1). One of the most highly regulated steps is the recruitment of mRNA, which requires recognition of the 5Ј-terminal m 7 GTP-containing cap and 3Ј-terminal poly(A) tract, unwinding of 5Ј-terminal secondary structure, and binding to the 43 S initiation complex. These steps are mediated by members of the eIF4 1 group of initiation factors (eIF4A, eIF4B, eIF4E, and eIF4G) as well as poly(A)-binding protein.eIF4A is the prototypical member of the DEXD/H-box protein family of nucleic acid helicases (2, 3). It functions as an ATPdependent, bi-directional RNA helicase and RNA-dependent ATPase (4 -7). The ␥-phosphate on the bound nucleotide has been shown to mediate changes in eIF4A conformation and RNA affinity. ATP binding and hydrolysis produce conformational changes in eIF4A that alter the RNA-protein interactions (8, 9, 3). A current model for protein synthesis initiation envisions eIF4A in the role of unwinding mRNA secondary structure in the 5Ј-untranslated region to allow the 40 S ribosomal subunit to bind the mRNA and/or scan it for the first AUG. The observation that dominant negative variants of eIF4A inhibit translation is consistent with such a role (10). Interestingly, the RNA helicase activity of eIF4A is ϳ20 times greater when bound to eIF4G than as a free protein (6, 11).There are at least two genes for eIF4G in humans (12-14), wheat germ (15), and yeast (16). In mammals, these are termed eIF4G-1 and eIF4G-2.2 eIF4G serves to colocalize initiation factors involved in mRNA recruitment to the 43 S initiation complex. It directly binds RNA (17-19), poly(A)-binding protein (13), eIF4E (20, 21), the 40 S-binding protein complex eIF3 (20), eIF4A (...

The eukaryotic translation factor 4A (eIF4A) is a member of DEA(D/H)-box RNA helicase family, a diverse group of proteins that couples ATP hydrolysis to RNA binding and duplex separation. eIF4A participates in the initiation of translation by unwinding secondary structure in the 5-untranslated region of mRNAs and facilitating scanning by the 40 S ribosomal subunit for the initiation codon. eIF4A alone has only weak ATPase and helicase activities, but these are stimulated by eIF4G, eIF4B, and eIF4H. eIF4G has two eIF4A-binding sites, one in the central domain (cp C3 ) and one in the COOH-terminal domain (cp C2 ). In the current work, we demonstrate that these two eIF4G domains have different effects on the RNA-stimulated ATPase activity of eIF4A. Initiation of translation is the most studied step for regulation of protein synthesis (1, 2). It involves the sequential assembly of initiation complex (43, 48, and 80 S), each step catalyzed by a different class of initiation factors (eIF1-eIF6) 1 (3). The rate-limiting step under normal conditions (i.e. absence of viral infection, amino acid starvation, chemical poisoning, etc.) is the recruitment of mRNA to the 43 S complex to form the 48 S complex, which is catalyzed by the eIF4 class of factors. The mammalian eIF4 proteins consist of eIF4A, a 46-kDa ATP-dependent RNA helicase; eIF4B, an 80-kDa protein that stimulates the processivity of eIF4A; eIF4H, a 25-kDa protein that enhances the stimulatory activity of eIF4B; eIF4E, a 25-kDa cap-binding protein; and eIF4G, a 185-kDa protein that binds most factors involved in mRNA recruitment and anchors them to the 40 S subunit through binding to eIF3. These factors are required both to recognize mRNA and to unwind secondary structure in the 5Ј-untranslated region, which is necessary for 48 S initiation complex formation and scanning for the initiation codon.Helicases unwind duplex DNA or RNA at the expense of energy derived from NTP hydrolysis (4). Sequence comparisons have classified the helicases into three superfamilies, SFI, SFII, and SFIII (5). There are striking similarities in the tertiary structure of two core domains called 1A and 2A (6, 7). These domains have homology with each other and also with the central region of the RecA protein (8). Domain 1A contains motifs I, Ia, Ic, II, and III, whereas domain 2A contains motifs IV, V, and VI. The most highly conserved motifs are Walker A and B, which possess sequences characteristic of ATPases (9). The ATP-binding site is situated in a cleft between domains 1A and 2A. The bound NTP acts as a cross-bridge pulling NH 2 -and COOH-terminal domains closer together. Conserved amino acid residues in both domains lining the active site cleft interact with the metal ion and the ␥-phosphate group of the bound NTP, thus stabilizing the transition state. Binding and hydrolysis of ATP at this site affect the relative positions of domains 1A and 2A.eIF4A is a bidirectional ATP-dependent helicase (10). It is the prototype for the DEA(D/H)-box protein family, which falls into SFII (4)...