Wheat eukaryotic initiation factor 4B organizes assembly of RNA and eIFiso4G, eIF4A, and poly(A)-binding protein

The eukaryotic translation initiation factor (eIF) 4B promotes the RNA-dependent ATP hydrolysis activity and ATP-dependent RNA helicase activity of eIF4A and eIF4F during translation initiation. Although this function is conserved among plants, animals, and yeast, eIF4B is one of the least conserved of initiation factors at the sequence level. To gain insight into its functional conservation, the organization of the functional domains of eIF4B from wheat has been investigated. Plant eIF4B contains three RNA binding domains, one more than reported for mammalian or yeast eIF4B, and each domain exhibits a preference for purine-rich RNA. In addition to a conserved RNA recognition motif and a C-terminal RNA binding domain, wheat eIF4B contains a novel N-terminal RNA binding domain that requires a short, lysine-rich containing sequence. Both the lysine-rich motif and an adjacent, C-proximal motif are conserved with an N-proximal sequence in human and yeast eIF4B. The C-proximal motif within the N-terminal RNA binding domain in wheat eIF4B is required for interaction with eIFiso4G, an interaction not reported for other eIF4B proteins. Moreover, each RNA binding domain requires dimerization for binding activity. Two binding sites for the poly(A)-binding protein were mapped to a region within each of two conserved 41-amino acid repeat domains on either side of the C-terminal RNA binding domain. eIF4A bound to an adjacent region within each repeat, supporting a central role for these conserved eIF4B domains in facilitating interaction with other components of the translational machinery. These results support the notion that eIF4B functions by organizing multiple components of the translation initiation machinery and RNA.     

The phosphorylation state of poly(A)-binding protein specifies its binding to poly(A) RNA and its interaction with eukaryotic initiation factor (eIF) 4F, eIFiso4F, and eIF4B

The poly(A)-binding protein (PABP) interacts with the eukaryotic initiation factor (eIF) 4G (or eIFiso4G), the large subunit of eIF4F (or eIFiso4F) to promote translation initiation. In plants, PABP also interacts with eIF4B, a factor that assists eIF4F function. PABP is a phosphoprotein, although the function of its phosphorylation has not been previously investigated. In this study, we have purified the phosphorylated and hypophosphorylated isoforms of PABP from wheat to examine whether its phosphorylation state affects its binding to poly(A) RNA and its interaction with eIF4G, eIFiso4G, or eIF4B. Phosphorylated PABP exhibited cooperative binding to poly(A) RNA even under non-stoichiometric binding conditions, whereas multiple molecules of hypophosphorylated PABP bound to poly(A) RNA only after free poly(A) RNA was no longer available. Together, phosphorylated and hypophosphorylated PABP exhibited synergistic binding. eIF4B interacted with PABP in a phosphorylation state-specific manner; native eIF4B increased the RNA binding activity specifically of phosphorylated PABP and was greater than 14-fold more effective than was recombinant eIF4B, whereas eIF4F promoted the cooperative binding of hypophosphorylated PABP. These data suggest that the phosphorylation state of PABP specifies the type of binding to poly(A) RNA and its interaction with its partner proteins.     

Translation initiation factor 4B homodimerization, RNA binding, and interaction with Poly(A)-binding protein are enhanced by zinc

The eukaryotic translation initiation factor (eIF) 4B promotes the RNA-dependent ATP hydrolysis activity and ATP-dependent RNA helicase activity of eIF4A and eIF4F during translation initiation. eIF4B also helps to organize the assembly of the translational machinery through its interactions with eIF4A, eIF4G, eIF3, the poly(A)-binding protein (PABP), and RNA. Although the function of eIF4B is conserved among plants, animals, and yeast, eIF4B is one of the least conserved of initiation factors at the sequence level. Mammalian eIF4B is a constitutive dimer; however, conflicting reports have suggested that plant eIF4B may exist as a monomer or a dimer. In this study, we show that eIF4B from wheat can form a dimer and we identify the region responsible for its dimerization. Zinc stimulated homodimerization of eIF4B and bound eIF4B with a Kd of 19.7 nM. Zinc increased the activity of the eIF4B C-terminal RNA-binding domain specifically. Zinc promoted the interaction between eIF4B and PABP but not the interaction between eIF4B and eIF4A or eIFiso4G, demonstrating that the effect of zinc was highly specific. The interaction between PABP and eIFiso4G was also stimulated by zinc but required significantly higher levels of zinc. Interestingly zinc abolished the ability of eIFiso4G to compete with eIF4B in binding to their overlapping binding sites in PABP by preferentially promoting the interaction between eIF4B and PABP. Our observations suggest that wheat eIF4B can dimerize but requires zinc. Moreover zinc controls the partner protein selection of PABP such that the interaction with eIF4B is preferred over eIFiso4G.     

Translation driven by an eIF4G core domain in vivo.

Most eukaryotic mRNAs possess a 5' cap structure (m(7)GpppN) and a 3' poly(A) tail which promote translation initiation by binding the eukaryotic translation initiation factor (eIF)4E and the poly(A) binding protein (PABP), respectively. eIF4G can bridge between eIF4E and PABP, and-through eIF3-is thought to establish a link to the small ribosomal subunit. We fused the C-terminal region of human eIF4GI lacking both the eIF4E- and PABP-binding sites, to the IRE binding protein IRP-1. This chimeric protein suffices to direct the translation of the downstream cistron of bicistronic mRNAs bearing IREs in their intercistronic space in vivo. This function is preserved even when translation via the 5' end is inhibited. Deletion analysis defined the conserved central domain (amino acids 642-1091) of eIF4G as an autonomous 'ribosome recruitment core' and implicated eIF4A as a critical binding partner. Our data reveal the sufficiency of the conserved eIF4G ribosome recruitment core to drive productive mRNA translation in living cells. The C-terminal third of eIF4G is dispensable, and may serve as a regulatory domain.     

Tobacco etch virus mRNA preferentially binds wheat germ eukaryotic initiation factor (eIF) 4G rather than eIFiso4G

The 5'-leader of tobacco etch virus (TEV) genomic RNA directs the efficient translation from the naturally uncapped viral RNA. The TEV 143-nt 5'-leader folds into a structure that contains two domains, each of which contains RNA pseudoknots. The 5'-proximal pseudoknot 1 (PK1) is necessary to promote cap-independent translation (Zeenko, V., and Gallie, D. R. (2005) J. Biol. Chem. 280, 26813-26824). During the translation initiation of cellular mRNAs, eIF4G functions as an adapter that recruits many of the factors involved in stimulating 40 S ribosomal subunit binding to an mRNA. Two related but highly distinct eIF4G proteins are expressed in plants, animals, and yeast. The two plant eIF4G isoforms, referred to as eIF4G and eIFiso4G, differ in size (165 and 86 kDa, respectively) and their functional differences are still unclear. Although eIF4G is required for the translation of TEV mRNA, it is not known if eIF4G binds directly to the TEV RNA itself or if other factors are required. To determine whether binding affinity and isoform preference correlates with translational efficiency, fluorescence spectroscopy was used to measure the binding of eIF4G, eIFiso4G, and their complexes (eIF4F and eIFiso4F, respectively) to the TEV 143-nt 5'-leader (TEV1-143) and a shorter RNA that contained PK1. A mutant (i.e. S1-3) in which the stem of PK1 was disrupted resulting in impaired cap-independent translation, was also tested. These studies demonstrate that eIF4G binds TEV1-143 and PK1 RNA with approximately 22-30-fold stronger affinity than eIFiso4G. eIF4G and eIF4F bind TEV1-143 with similar affinity, whereas eIFiso4F binds with approximately 6-fold higher affinity than eIFiso4G. The binding affinity of eIF4G, eIF4F, and eIFiso4G to S1-3 was reduced by 3-5-fold, consistent with the reduction in the ability of this mutant to promote cap-independent translation. Temperature-dependent binding studies revealed that binding of the TEV 5'-leader to these initiation factors has a large entropic contribution. Overall, these results demonstrate the first direct interaction of eIF4G with the TEV 5'-leader in the absence of other initiation factors. These data correlate well with the observed translational data and provide more detailed information on the translational strategy of potyviruses.     

Effects of poly(A)-binding protein on the interactions of translation initiation factor eIF4F and eIF4F.4B with internal ribosome entry site (IRES) of tobacco etch virus RNA

In wheat germ, the interaction between poly(A)-binding protein and eukaryotic initiation factor eIF 4G increases the affinity of eIF4E for the cap by 20-40-fold. Recent findings that wheat germ eIF4G is required for interaction with the IRES, pseudoknot 1 (PK1), of tobacco etch virus to promote cap-independent translation led us to investigate the effects of PABP on the interaction of eIF4F with PK1. The fluorescence anisotropy data showed addition of PABP to eIF4F increased the binding affinity approximately 2.0-fold for PK1 RNA as compared with eIF4F alone. Addition of both PABP and eIF4B to eIF4F enhance binding affinity to PK1 about 4-fold, showing an additive effect rather than the large increase in affinity shown for cap binding. The van't Hoff analyses showed that PK1 RNA binding to eIF4F, eIF4F.PABP, eIF4F.4B and eIF4F.4B.PABP is enthalpy-driven and entropy-favorable. PABP and eIF4B decreased the entropic contribution 65% for binding of PK1 RNA to eIF4F. The lowering of entropy for the formation of eIF4F.4B.PABP-PK1 complex suggested reduced hydrophobic interactions for complex formation. Overall, these results demonstrate the first direct effect of PABP on the interaction of eIF4F and eIF4F.4B with PK1 RNA.     

Translation initiation factor (eIF) 4B affects the rates of binding of the mRNA m7G cap analogue to wheat germ eIFiso4F and eIFiso4F.PABP

Previous kinetic binding studies of wheat germ protein synthesis eukaryotic translational initiation factor eIFiso4F and its subunit, eIFiso4E, with m(7)GTP and mRNA analogues indicated that binding occurred by a two-step process with the first step occurring at a rate close to the diffusion-controlled rate [Sha, M., Wang, Y., Xiang, T., van Heerden, A., Browning, K. S., and Goss, D. J. (1995) J. Biol. Chem. 270, 29904-29909]. The kinetic effects of eIF4B, PABP, and wheat germ eIFiso4F with two mRNA cap analogues and the temperature dependence of this reaction were measured and compared. The Arrhenius activation energies for binding of the two mRNA cap analogues, Ant-m(7)GTP and m(7)GpppG, were significantly different. Fluorescence stopped-flow studies of the eIFiso4F.eIF4B protein complex with two m(7)G cap analogues show a concentration-independent conformational change. The rate of this conformational change was approximately 2.4-fold faster for the eIFiso4F.eIF4B complex compared with our previous studies of eIFiso4F [Sha, M., Wang, Y., Xiang, T., van Heerden, A., Browning, K. S., and Goss, D. J. (1995) J. Biol. Chem. 270, 29904-29909]. The dissociation rates were 3.7- and 5.4-fold slower for eIFiso4F.Ant-m(7)GTP and eIFiso4F.m(7)GpppG, respectively, in the presence of eIF4B and PABP. These studies show that eIF4B and PABP enhance the interaction with the cap and probably are involved in protein-protein interactions as well. The temperature dependence of the cap binding reaction was markedly reduced in the presence of either eIF4B or PABP. However, when both eIF4B and PABP were present, not only was the energy barrier reduced but the binding rate was faster. Since cap binding is thought to be the rate-limiting step in protein synthesis, these two proteins may perform a critical function in regulation of the overall protein synthesis efficiency. This suggests that the presence of both proteins leads to a rapid, stable complex, which serves as a scaffold for further initiation complex formation.     

Two structurally atypical HEAT domains in the C-terminal portion of human eIF4G support binding to eIF4A and Mnk1

The X-ray structure of the C-terminal region of human eukaryotic translation initiation factor 4G (eIF4G) has been determined at 2.2 A resolution, revealing two atypical HEAT-repeat domains. eIF4G recruits various translation factors and the 40S ribosomal subunit to the mRNA 5' end. In higher eukaryotes, the C terminus of eIF4G (4G/C) supports translational regulation by recruiting eIF4A, an RNA helicase, and Mnk1, the kinase responsible for phosphorylating eIF4E. Structure-guided surface mutagenesis and protein-protein interaction assays were used to identify binding sites for eIF4A and Mnk1 within the HEAT-repeats of 4G/C. p97/DAP5, a translational modulator homologous to eIF4G, lacks an eIF4A binding site in the corresponding region. The second atypical HEAT domain of the 4G/C binds Mnk1 using two conserved aromatic/acidic-box (AA-box) motifs. Within the first AA-box, the aromatic residues contribute to the hydrophobic core of the domain, while the acidic residues form a negatively charged surface feature suitable for electrostatic interactions with basic residues in Mnk1.     

Recognition of eIF4G by rotavirus NSP3 reveals a basis for mRNA circularization

Rotaviruses, segmented double-stranded RNA viruses, co-opt the eukaryotic translation machinery with the aid of nonstructural protein 3 (NSP3), a rotaviral functional homolog of the cellular poly(A) binding protein (PABP). NSP3 binds to viral mRNA 3' consensus sequences and circularizes mRNA via interactions with eIF4G. Here, we present the X-ray structure of the C-terminal domain of NSP3 (NSP3-C) recognizing a fragment of eIF4GI. Homodimerization of NSP3-C yields a symmetric, elongated, largely alpha-helical structure with two hydrophobic eIF4G binding pockets at the dimer interface. Site-directed mutagenesis and isothermal titration calorimetry documented that NSP3 and PABP use analogous eIF4G recognition strategies, despite marked differences in tertiary structure.     

Wheat germ poly(A)-binding protein increases the ATPase and the RNA helicase activity of translation initiation factors eIF4A, eIF4B, and eIF-iso4F

Recent studies demonstrated that wheat germ poly(A)-binding protein (PABP) interacted with translation eukaryotic initiation factor (eIF)-iso4G and eIF4B, and these interactions increased the poly(A) binding activity of PABP (Le, H., Tanguay, R. L., Balasta, M. L., Wei, C. C., Browning, K. S., Metz, A. M., Goss, D. J., and Gallie, D. R. (1997) J. Biol. Chem. 272, 16247-16255) and the cap binding activity of eIF-iso4F (Wei, C. C., Balasta, M. L., Ren, J., and Goss, D. J. (1998) Biochemistry 37, 1910-1916). We report here that the interaction between PABP and eIF-iso4G has a substantial effect on the ATPase activity and RNA helicase activity of (eIF4A + eIF4B + eIF-iso4F) complex. ATPase kinetic assays show, in the presence of poly(U), PABP can increase the parameter (k(cat)/K(m)) by 3.5-fold with a 2-fold decrease of K(m) for the (eIF4A + eIF-iso4F) complex. In the presence of globin messenger RNA, the ATPase activity of the complex (eIF4A + eIF-iso4F) was increased 2-fold by the presence of PABP. RNA helicase assays demonstrated that the presence of PABP enhanced the RNA duplex unwinding activity of the initiation factor complex. These results suggest that, in terms of the scanning model of translation initiation, PABP may enhance the mRNA scanning rate of the complex formed by eIF4A, eIF4B, and eIF4F or eIF-(iso)4F and increase the rate of translation.     

Identification of a nucleic acid binding domain in eukaryotic initiation factor eIFiso4G from wheat

Higher plants have two complexes that bind the m7G-cap structure of mRNA and mediate interactions between mRNA and ribosomal subunits, designated eIF4F and eIFiso4F. Both complexes contain a small subunit that binds the 5'-cap structure of mRNA, and a large subunit, eIF4G or eIFiso4G, that binds other translation factors and RNA. Sequence-specific proteases were used to cleave native cap-binding complexes into structural domains, which were purified by affinity chromatography. We show here that eIFiso4G contains a central protease-resistant domain that binds specifically to nucleic acids. This domain spans Gln170 to Glu443 and includes four of the six homology blocks shared by eIFiso4G and eIF4G. A slightly shorter overlapping sequence, from Gly202 to Lys445, had no nucleic acid binding activity, indicating that the N-terminal end of the nucleic acid binding site lies within Gln170 to Arg201. The binding of the central domain and native eIFiso4F to RNA homopolymers and double- and single-stranded DNAs was studied. Both molecules had highest affinity for poly(G) and recognized single- and double-stranded sequences.     

A newly identified N-terminal amino acid sequence of human eIF4G binds poly(A)-binding protein and functions in poly(A)-dependent translation.

Most eukaryotic mRNAs possess a 5' cap and a 3' poly(A) tail, both of which are required for efficient translation. In yeast and plants, binding of eIF4G to poly(A)-binding protein (PABP) was implicated in poly(A)-dependent translation. In mammals, however, there has been no evidence that eIF4G binds PABP. Using 5' rapid amplification of cDNA, we have extended the known human eIF4GI open reading frame from the N-terminus by 156 amino acids. Co-immunoprecipitation experiments showed that the extended eIF4GI binds PABP, while the N-terminally truncated original eIF4GI cannot. Deletion analysis identified a 29 amino acid sequence in the new N-terminal region as the PABP-binding site. The 29 amino acid stretch is almost identical in eIF4GI and eIF4GII, and the full-length eIF4GII also binds PABP. As previously shown for yeast, human eIF4G binds to a fragment composed of RRM1 and RRM2 of PABP. In an in vitro translation system, an N-terminal fragment which includes the PABP-binding site inhibits poly(A)-dependent translation, but has no effect on translation of a deadenylated mRNA. These results indicate that, in addition to a recently identified mammalian PABP-binding protein, PAIP-1, eIF4G binds PABP and probably functions in poly(A)-dependent translation in mammalian cells.     

The RNA recognition motif of yeast translation initiation factor Tif3/eIF4B is required but not sufficient for RNA strand-exchange and translational activity.

The Saccharomyces cerevisiae TIF3 gene encodes a 436-amino acid (aa) protein that is the yeast homologue of mammalian translation Initiation factor eIF4B. Tif3p can be divided into three parts, the N-terminal region with an RNA recognition motif (RRM) (aa 1-182), followed in the middle part by a sevenfold repeat of 26 amino acids rich in basic and acidic residues (as 183-350), and a C-terminal region without homology to any known sequence (aa 351-436). We have analyzed several Tif3 proteins with deletions at their N and C termini for their ability (1) to complement a tif3delta strain in vivo, (2) to stimulate Tif3-dependent translation extracts, (3) to bind to single-stranded RNA, and (4) to catalyze RNA strand-exchange in vitro. Here we report that yeast Tif3/eIF4B contains at least two RNA binding domains able to bind to single-stranded RNA. One is located in the N-terminal region of the protein carrying the RRM, the other in the C-terminal two-thirds region of Tif3p. The RRM-containing domain and three of the seven repeat motifs are essential for RNA strand-exchange activity of Tif3p and translation in vitro and for complementation of a tif3delta strain, suggesting an important role for RNA strand-exchange activity in translation.     

Wheat germ translation initiation factor eIF4B affects eIF4A and eIFiso4F helicase activity by increasing the ATP binding affinity of eIF4A

It has been proposed that, during translational initiation, structures in the 5' untranslated region of mRNA are unwound. eIF4A, a member of the DEAD box family of proteins (those that contain a DEAD amino acid sequence), separately or in conjunction with other eukaryotic initiation factors, utilizes the energy from ATP hydrolysis to unwind these structures. As a step in defining the mechanism of helicase activity in the wheat germ protein synthesis system, we have utilized direct fluorescence measurements, ATPase assays, and helicase assays. The RNA duplex unwinding activity of wheat germ eIF4A is similar to other mammalian systems; however, eIF4F or eIFiso4F is required, probably because of the low binding affinity of wheat germ eIF4A for mRNA. Direct ATP binding measurements showed that eIF4A had a higher binding affinity for ADP than ATP, resulting in a limited hydrolysis and procession along the RNA in the helicase assay. The addition of eIF4B resulted in a change in binding affinity for ATP, increasing it almost 10-fold while the ADP binding affinity was approximately the same. The data presented in this paper suggest that eIF4F or eIFiso4F acts to position the eIF4A and stabilize the interaction with mRNA. ATP produces a conformational change which allows a limited unwinding of the RNA duplex. The binding of eIF4B either prior to or after hydrolysis allows for increased affinity for ATP and for the cycle of conformational changes to proceed, resulting in further unwinding and processive movement along the mRNA.     

Physical association of eukaryotic initiation factor 4G (eIF4G) with eIF4A strongly enhances binding of eIF4G to the internal ribosomal entry site of encephalomyocarditis virus and is required for internal initiation of translation

Mammalian eukaryotic initiation factor 4GI (eIF4GI) may be divided into three similarly sized regions. The central region (amino acids [aa] 613 to 1090) binds eIF3, eIF4A, and the encephalomyocarditis virus (EMCV) internal ribosomal entry site (IRES) and mediates initiation on this RNA. We identified the regions of eIF4GI that are responsible for its specific interaction with the IRES and that are required to mediate 48S complex formation on the IRES in vitro. Mutational analysis demarcated the IRES binding fragment of eIF4GI (aa 746 to 949) and indicated that it does not resemble an RNA recognition motif (RRM)-like domain. An additional amino-terminal sequence (aa 722 to 746) was required for binding eIF4A and for 48S complex formation. eIF4GI bound the EMCV IRES and beta-globin mRNA with similar affinities, but association with eIF4A increased its affinity for the EMCV IRES (but not beta-globin RNA) by 2 orders of magnitude. On the other hand, eIF4GI mutants with defects in binding eIF4A were defective in mediating 48S complex formation even if they bound the IRES normally. These data indicate that the eIF4G-eIF4A complex, rather than eIF4G alone, is required for specific high-affinity binding to the EMCV IRES and for internal ribosomal entry on this RNA.     

Mitotic Phosphorylation of Eukaryotic Initiation Factor 4G1 (eIF4G1) at Ser1232 by Cdk1:Cyclin B Inhibits eIF4A Helicase Complex Binding with RNA

During mitosis, global translation is suppressed, while synthesis of proteins with vital mitotic roles must go on. Prior evidence suggests that the mitotic translation shift involves control of initiation. Yet, no signals specifically targeting translation initiation factors during mitosis have been identified. We used phosphoproteomics to investigate the central translation initiation scaffold and “ribosome adaptor,” eukaryotic initiation factor 4G1 (eIF4G1) in interphase or nocodazole-arrested mitotic cells. This approach and kinase inhibition assays, in vitro phosphorylation with recombinant kinase, and kinase depletion-reconstitution experiments revealed that Ser1232 in eIF4G1 is phosphorylated by cyclin-dependent kinase 1 (Cdk1):cyclin B during mitosis. Ser1232 is located in an unstructured region of the C-terminal portion of eIF4G1 that coordinates assembly of the eIF4G/-4A/-4B helicase complex and binding of the mitogen-activated protein kinase (MAPK) signal-integrating kinase, Mnk. Intense phosphorylation of Ser1232 in mitosis strongly enhanced the interactions of eIF4A with HEAT domain 2 of eIF4G and decreased association of eIF4G/-4A with RNA. Our findings implicate phosphorylation of eIF4G1(Ser1232) by Cdk1:cyclin B and its inhibitory effects on eIF4A helicase activity in the mitotic translation initiation shift.     

IRES interaction with translation initiation factors: functional characterization of novel RNA contacts with eIF3, eIF4B, and eIF4GII

Translation initiation promoted by picornavirus internal ribosome entry site (IRES) elements is dependent on the association of specific IRES sequences to the initiation factor eIF4G. However the RNA determinants interacting with other components of the translational machinery are still unknown. In this study, we have identified novel RNA-protein interactions between the foot-and-mouth disease virus (FMDV) IRES and three translation initiation factors. A doublet of 116/110 kDa that crosslinked to the FMDV IRES is a component of eIF3. We show here that domain 5 holds the preferential binding site for eIF3, although this complex initiation factor can establish multiple contacts with the IRES structure. We have also identified the phylogenetically conserved hairpin of domain 5 as the RNA motif responsible for eIF4B interaction. Mutation of this stem-loop structure abrogated eIF4B, but not eIF3, binding to the IRES. Remarkably, IRES mutants severely affected in their interaction with eIF4B showed a mild reduction in IRES activity when tested in the context of a bicistronic expression vector in transfected cells. Finally, we provide evidence of the interaction of eIF4GII with FMDV IRES, the RNA determinants for this interaction being shared with its functional homolog eIF4GI. The FMDV Lb protease generated a C-terminal fragment of eIF4GII that binds to the IRES as efficiently as the intact protein. Competition experiments showed that titration of eIF4B or p110/116 interaction with the FMDV IRES required a large excess of competitor relative to eIF4G, strongly suggesting that eIF4G-IRES interaction is a limiting factor to titrate the IRES. Comparative analysis of the activity of IRES mutants affected in domains 4 and 5 regarding their pattern of RNA-protein complex formation demonstrates that while binding of eIF4B with the FMDV IRES is dispensable, interaction of eIF4G is a central feature of the activity of this element.     

Paip1 interacts with poly(A) binding protein through two independent binding motifs

The 3' poly(A) tail of eukaryotic mRNAs plays an important role in the regulation of translation. The poly(A) binding protein (PABP) interacts with eukaryotic initiation factor 4G (eIF4G), a component of the eIF4F complex, which binds to the 5' cap structure. The PABP-eIF4G interaction brings about the circularization of the mRNA by joining its 5' and 3' termini, thereby stimulating mRNA translation. The activity of PABP is regulated by two interacting proteins, Paip1 and Paip2. To study the mechanism of the Paip1-PABP interaction, far-Western, glutathione S-transferase pull-down, and surface plasmon resonance experiments were performed. Paip1 contains two binding sites for PABP, PAM1 and PAM2 (for PABP-interacting motifs 1 and 2). PAM2 consists of a 15-amino-acid stretch residing in the N terminus, and PAM1 encompasses a larger C-terminal acidic-amino-acid-rich region. PABP also contains two Paip1 binding sites, one located in RNA recognition motifs 1 and 2 and the other located in the C-terminal domain. Paip1 binds to PABP with a 1:1 stoichiometry and an apparent K(d) of 1.9 nM.     

Interaction between the NH2-terminal domain of eIF4A and the central domain of eIF4G modulates RNA-stimulated ATPase activity

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. cp(C3) stimulates ATP-hydrolytic efficiency by about 40-fold through two mechanisms: lowering K(m)(RNA) by 10-fold and raising k(cat) by 4-fold. cp(C3) also stimulates RNA cross-linking to eIF4A in an ATP-independent manner. Studies with eIF4G and eIF4A variants suggest a model by which cp(C3) alters the conformation of the catalytic site to favor RNA binding. cp(C2) does not stimulate ATPase activity and furthermore increases both K(m)(ATP) (at saturating RNA concentrations) and K(m)(RNA) (at subsaturating ATP concentrations). Both cp(C3) and cp(C2) directly interact with the NH(2)-terminal domain of eIF4A, which possesses conserved ATP- and oligonucleotide-binding motifs, but not with the COOH-terminal domain.     

The yeast eukaryotic initiation factor 4G (eIF4G) HEAT domain interacts with eIF1 and eIF5 and is involved in stringent AUG selection

Eukaryotic initiation factor 4G (eIF4G) promotes mRNA recruitment to the ribosome by binding to the mRNA cap- and poly(A) tail-binding proteins eIF4E and Pap1p. eIF4G also binds eIF4A at a distinct HEAT domain composed of five stacks of antiparallel alpha-helices. The role of eIF4G in the later steps of initiation, such as scanning and AUG recognition, has not been defined. Here we show that the entire HEAT domain and flanking residues of Saccharomyces cerevisiae eIF4G2 are required for the optimal interaction with the AUG recognition factors eIF5 and eIF1. eIF1 binds simultaneously to eIF4G and eIF3c in vitro, as shown previously for the C-terminal domain of eIF5. In vivo, co-overexpression of eIF1 or eIF5 reverses the genetic suppression of an eIF4G HEAT domain Ts(-) mutation by eIF4A overexpression. In addition, excess eIF1 inhibits growth of a second eIF4G mutant defective in eIF4E binding, which was also reversed by co-overexpression of eIF4A. Interestingly, excess eIF1 carrying the sui1-1 mutation, known to relax the accuracy of start site selection, did not inhibit the growth of the eIF4G mutant, and sui1-1 reduced the interaction between eIF4G and eIF1 in vitro. Moreover, a HEAT domain mutation altering eIF4G moderately enhances translation from a non-AUG codon. These results strongly suggest that the binding of the eIF4G HEAT domain to eIF1 and eIF5 is important for maintaining the integrity of the scanning ribosomal preinitiation complex.