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.     

Poly(A)-binding protein increases the binding affinity and kinetic rates of interaction of viral protein linked to genome with translation initiation factors eIFiso4F and eIFiso4F·4B complex

VPg of turnip mosaic virus (TuMV) was previously shown to interact with translation initiation factor eIFiso4F and play an important role in mRNA translation [Khan, M. A., et al. (2008) J. Biol. Chem.283, 1340-1349]. VPg competed with cap analogue for eIFiso4F binding and competitively inhibited cap-dependent translation and enhanced cap-independent translation to give viral RNA a significant competitive advantage. To gain further insight into the cap-independent process of initiation of protein synthesis, we examined the effect of PABP and/or eIF4B on the equilibrium and kinetics of binding of VPg to eIFiso4F. Equilibrium data showed the addition of PABP and/or eIF4B to eIFiso4F increased the binding affinity for VPg (K(d) = 24.3 ± 1.6 nM) as compared to that with eIFiso4F alone (K(d) = 81.3 ± 0.2.4 nM). Thermodynamic parameters showed that binding of VPg to eIFiso4F was enthalpy-driven and entropy-favorable with the addition of PABP and/or eIF4B. PABP and eIF4B decreased the entropic contribution by 67% for binding of VPg to eIFiso4F. The decrease in entropy involved in the formation of the eIFiso4F·4B·PABP-VPg complex suggested weakened hydrophobic interactions for complex formation and an overall conformational change. The kinetic studies of eIFiso4F with VPg in the presence of PABP and eIF4B show 3-fold faster association (k(2) = 182 ± 9.0 s(-1)) compared to that with eIFiso4F alone (k(2) = 69.0 ± 1.5 s(-1)) . The dissociation rate was 3-fold slower (k(-2) = 6.5 ± 0.43 s(-1)) for eIFiso4F with VPg in the presence of PABP and eIF4B (k(-2) = 19.0 ± 0.9 s(-1)). The addition of PABP and eIF4B decreased the activation energy of eIFiso4F with VPg from 81.0 ± 3.0 to 44.0 ± 2.4 kJ/mol. This suggests that the presence of both proteins leads to a rapid, stable complex, which serves to sequester initiation factors.     

Interaction of genome-linked protein (VPg) of turnip mosaic virus with wheat germ translation initiation factors eIFiso4E and eIFiso4F

The interaction between VPg of turnip mosaic virus and wheat germ eukaryotic translation initiation factors eIFiso4E and eIFiso4F (the complex of eIFiso4E and eIFiso4G) were measured and compared. The fluorescence quenching data showed the presence of one binding site on eIFiso4E for VPg. Scatchard analysis revealed the binding affinity (K(a)) and average binding sites (n) for VPg were (8.51 +/- 0.21) x 10(6) M(-1) and 1.0, respectively. The addition of eIFiso4G to the eIFiso4E increased the binding affinity 1.5-fold for VPg as compared with eIFiso4E alone. However, eIFiso4G alone did not bind with VPg. The van't Hoff analyses showed that VPg binding is enthalpy-driven and entropy-favorable with a large negative DeltaH degrees (-29.32 +/- 0.13 kJmol(-1)) and positive DeltaS degrees (36.88 +/- 0.25 Jmol(-1)K(-1)). A Lineweaver-Burk plot indicates mixed-type competitive ligand binding between VPg and anthraniloyl-7-methylguanosine triphosphate for eIFiso4E. Fluorescence stopped-flow studies of eIFiso4E and eIFiso4F with VPg show rapid binding, suggesting kinetic competition between VPg and m(7)G cap. The VPg protein binds much faster than cap analogs. The activation energies for binding of eIFiso4E and eIFiso4F with VPg were 50.70 +/- 1.27 and 75.37 +/- 2.95 kJmol(-1) respectively. Enhancement of eIFiso4F-VPg binding with the addition of a structured RNA derived from tobacco etch virus suggests that translation initiation involving VPg occurs at internal ribosomal entry sites. Furthermore, the formation of a protein-RNA complex containing VPg suggests the possibility of direct participation of VPg in the translation of the viral genome.     

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.     

Potyvirus genome-linked protein, VPg, directly affects wheat germ in vitro translation: interactions with translation initiation factors eIF4F and eIFiso4F

Potyvirus genome linked protein, VPg, interacts with translation initiation factors eIF4E and eIFiso4E, but its role in protein synthesis has not been elucidated. We show that addition of VPg to wheat germ extract leads to enhancement of uncapped viral mRNA translation and inhibition of capped viral mRNA translation. This provides a significant competitive advantage to the uncapped viral mRNA. To understand the molecular basis of these effects, we have characterized the interaction of VPg with eIF4F, eIFiso4F, and a structured RNA derived from tobacco etch virus (TEV RNA). When VPg formed a complex with eIF4F, the affinity for TEV RNA increased more than 4-fold compared with eIF4F alone (19.4 and 79.0 nm, respectively). The binding affinity of eIF4F to TEV RNA correlates with translation efficiency. VPg enhanced eIFiso4F binding to TEV RNA 1.6-fold (178 nm compared with 108 nm). Kinetic studies of eIF4F and eIFiso4F with VPg show approximately 2.6-fold faster association for eIFiso4F.VPg as compared with eIF4F.VPg. The dissociation rate was approximately 2.9-fold slower for eIFiso4F than eIF4F with VPg. These data demonstrate that eIFiso4F can kinetically compete with eIF4F for VPg binding. The quantitative data presented here suggest a model where eIF4F.VPg interaction enhances cap-independent translation by increasing the affinity of eIF4F for TEV RNA. This is the first evidence of direct participation of VPg in translation initiation.     

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.     

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.     

Characterization of pokeweed antiviral protein binding to mRNA cap analogs: competition with nucleotides and enhancement by translation initiation factor iso4G

Pokeweed antiviral protein (PAP) is a type I ribosomal inactivating protein (RIP). PAP binds to and depurinates the sarcin/ricin loop (SRL) of ribosomal RNA resulting in the cessation of protein synthesis. PAP has also been shown to bind to mRNA cap analogs and depurinate mRNA downstream of the cap structure. The biological role of cap binding and its possible role in PAP activity are not known. Here we show the first direct quantitative evidence for PAP binding to the cap analog m(7)GTP. We report a binding affinity of 43.3+/-0.1 nM at 25 degrees C as determined by fluorescence quenching experiments. This is similar to the values reported for wheat cap-binding proteins eIFiso4E and eIFiso4F. van't Hoff analysis of m(7)GTP-PAP equilibrium reveals a binding reaction that is enthalpy driven and entropy favored with TDeltaS degrees contributing 15% to the overall value of DeltaG degrees . This is in contrast to the wheat cap-binding proteins which are enthalpically driven in the DeltaG degrees for binding. Competition experiments indicate that ATP and GTP compete for the cap-binding site on PAP with slightly different affinities. Fluorescence studies of PAP-eIFiso4G binding reveal a protein-protein interaction with a K(d) of 108.4+/-0.3 nM. eIFiso4G was shown to enhance the interaction of PAP with m(7)GTP cap analog by 2.4-fold. These results suggest the involvement of PAP-translation initiation factor complexes in RNA selection and depurination.     

Plant cap-binding complexes eukaryotic initiation factors eIF4F and eIFISO4F: molecular specificity of subunit binding

The initiation of translation in eukaryotes requires a suite of eIFs that include the cap-binding complex, eIF4F. eIF4F is comprised of the subunits eIF4G and eIF4E and often the helicase, eIF4A. The eIF4G subunit serves as an assembly point for other initiation factors, whereas eIF4E binds to the 7-methyl guanosine cap of mRNA. Plants have an isozyme form of eIF4F (eIFiso4F) with comparable subunits, eIFiso4E and eIFiso4G. Plant eIF4A is very loosely associated with the plant cap-binding complexes. The specificity of interaction of the individual subunits of the two complexes was previously unknown. To address this issue, mixed complexes (eIF4E-eIFiso4G or eIFiso4E-eIF4G) were expressed and purified from Escherichia coli for biochemical analysis. The activity of the mixed complexes in in vitro translation assays correlated with the large subunit of the respective correct complex. These results suggest that the eIF4G or eIFiso4G subunits influence translational efficiency more than the cap-binding subunits. The translation assays also showed varying responses of the mRNA templates to eIF4F or eIFiso4F, suggesting that some level of mRNA discrimination is possible. The dissociation constants for the correct complexes have K(D) values in the subnanomolar range, whereas the mixed complexes were found to have K(D) values in the ～10 nm range. Displacement assays showed that the correct binding partner readily displaces the incorrect binding partner in a manner consistent with the difference in K(D) values. These results show molecular specificity for the formation of plant eIF4F and eIFiso4F complexes and suggest a role in mRNA discrimination during initiation of translation.     

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.     

eIF4G, eIFiso4G, and eIF4B bind the poly(A)-binding protein through overlapping sites within the RNA recognition motif domains

The poly(A)-binding protein (PABP), a protein that contains four conserved RNA recognition motifs (RRM1-4) and a C-terminal domain, is expressed throughout the eukaryotic kingdom and promotes translation through physical and functional interactions with eukaryotic initiation factor (eIF) 4G and eIF4B. Two highly divergent isoforms of eIF4G, known as eIF4G and eIFiso4G, are expressed in plants. As little is known about how PABP can interact with RNA and three distinct translation initiation factors in plants, the RNA binding specificity and organization of the protein interaction domains in wheat PABP was investigated. Wheat PABP differs from animal PABP in that its RRM1 does not bind RNA as an individual domain and that RRM 2, 3, and 4 exhibit different RNA binding specificities to non-poly(A) sequences. The PABP interaction domains for eIF4G and eIFiso4G were distinct despite the functional similarity between the eIF4G proteins. A single interaction domain for eIF4G is present in the RRM1 of PABP, whereas eIFiso4G interacts at two sites, i.e. one within RRM1-2 and the second within RRM3-4. The eIFiso4G binding site in RRM1-2 mapped to a 36-amino acid region encompassing the C-terminal end of RRM1, the linker region, and the N-terminal end of RRM2, whereas the second site in RRM3-4 was more complex. A single interaction domain for eIF4B is present within a 32-amino acid region representing the C-terminal end of RRM1 of PABP that overlaps with the N-proximal eIFiso4G interaction domain. eIF4B and eIFiso4G exhibited competitive binding to PABP, supporting the overlapping nature of their interaction domains. These results support the notion that eIF4G, eIFiso4G, and eIF4B interact with distinct molecules of PABP to increase the stability of the interaction between the termini of an mRNA.     

Translational control by the poly(A) binding protein: a check for mRNA integrity

The eukaryotic mRNA 3' poly(A) tail and the 5' cap cooperate to synergistically enhance translation. This interaction is mediated by a ribonucleoprotein network that contains, at a minimum, the poly(A) binding protein (PABP), the capbinding protein eIF4E and a scaffolding protein, eIF4G. eIF4G, in turn, contains binding sites for eIF4A and eIF3, a 40S ribosome-associated initiation factor. The combined cooperative interactions within this "closed loop" mRNP among other effects enhance the affinity of eIF4E for the 5' cap by lowering its dissociation rate and, ultimately, facilitate the formation of 48S and 80S ribosome initiation complexes. The PABP-poly(A) interaction also stimulates initiation driven by picomavirus' internal ribosomal entry sites (IRESs), a process that requires eIF4G but not eIF4E. PABP, therefore, should be considered a canonical initiation factor, integral to initiation complex formation. Poly(A)-mediated translation is subjected to regulation by the PABP-interacting proteins Paip1 and Paip2. Paip1 acts as a translational enhancer. In contrast, Paip2 strongly inhibits translation by promoting dissociation of PABP from poly(A) and by competing with eIF4G for binding to PABP.     

Poly(A) tail affects equilibrium and thermodynamic behavior of tobacco etch virus mRNA with translation initiation factors eIF4F,eIF4B and PABP

We have investigated the effects of poly(A)-tail on binding of eIF4F, eIF4B and PABP with tobacco etch virus (TEV) IRES RNA. The fluorescence anisotropy data showed that the addition of poly(A)₂₀ increases the binding affinity of eIF4F·4B and eIF4F·PABP complexes to IRES RNA ~ 2- and 4-fold, respectively. However, the binding affinity of eIF4F with PK1 was enhanced ~ 11-fold with the addition of PABP, eIF4B, and poly(A)₂₀ together. Whereas, poly(A)₂₀ alone increases the binding affinity of eIF4F·4B·PABP with PK1 RNA about 3-fold, showing an additive effect rather than the large increase in affinity as shown for cap binding. Thermodynamic data showed that PK1 RNA binding to protein complexes in the presence of poly(A)₂₀ was enthalpy-driven and entropy-favorable. Poly(A)₂₀ decreased the entropic contribution 75% for binding of PK1 RNA to eIF4F·4B·PABP as compared to eIF4F alone, suggesting reduced hydrophobic interactions for complex formation and an overall conformational change. Overall, these results demonstrate the first direct effect of poly(A) on the equilibrium and thermodynamics of eIF4F and eIF4F·4B·PABP with IRES-RNA.     

Kinetic Mechanism for the Binding of eIF4F and Tobacco Etch Virus Internal Ribosome Entry Site RNA: EFFECTS OF eIF4B AND POLY(A)-BINDING PROTEIN*

The wheat germ eukaryotic translation initiation factor (eIF) 4F binds tightly to the mRNA internal ribosome entry site (IRES) of tobacco etch virus (TEV) to promote translation initiation. When eIF4F is limiting, TEV is preferentially translated compared with host cell mRNA. To gain insight into the dynamic process of protein synthesis initiation and the mechanism of binding, the kinetics of eIF4F binding to TEV IRES were examined. The association rate constant (k_on) and dissociation rate constant (k_off) for eIF4F binding to IRES were 59 ± 2.1 μm⁻¹ s⁻¹ and 12.9 ± 0.3 s⁻¹, respectively, comparable with the rates for capped RNA. Binding of eIF4E or eIF4F to the cap of mRNA is the rate-limiting step for initiation of cap-dependent protein synthesis. The concentration dependence of the reactions suggested a simple one-step association mechanism. However, the association rate was reduced more than 10-fold when KCl concentration was increased from 50 to 300 mm, whereas the dissociation rate constant was increased 2-fold. The addition of eIF4B and poly(A)-binding protein enhanced the association rate of eIF4F ∼3-fold. These results suggest a mechanism where eIF4F initially binds electrostatically, followed by a conformational change to further stabilize binding. Poly(A)-binding protein and eIF4B mainly affect the eIF4F/TEV association rate. These results demonstrate the first direct kinetic measurements of translation initiation factor binding to an IRES.     

Translational control by the poly(A) binding protein: A check for mRNA integrity

The eukaryotic mRNA 3′ poly(A) tail and the 5′ cap cooperate to synergistically enhance translation. This interaction is mediated by a ribonucleoprotein network that contains, at a minimum, the poly(A) binding protein (PABP), the cap-binding protein eIF4E, and a scaffolding protein, eIF4G. eIF4G, in turn, contains binding sites for eIF4A and eIF3, a 40S ribosome-associated initiation factor. The combined cooperative interactions within this “closed loop” mRNA among other effects enhance the affinity of eIF4E for the 5′ cap, by lowering its dissociation rate and, ultimately, facilitate the formation of 48S and 80S ribosome initiation complexes. The PABP-poly(A) interaction also stimulates initiation driven by picornavirus’ internal ribosomal entry sites (IRESs), a process that requires eIF4G but not eIF4E. PABP, therefore, should be considered a canonical initiation factor, integral to the formation of the initiation complex. Poly(A)-mediated translation is subjected to regulation by the PABP-interacting proteins Paip1 and Paip2. Paip1 acts as a translational enhancer. In contrast, Paip2 strongly inhibits translation by promoting dissociation of PABP from poly(A) and by competing with eIF4G for binding to PABP. Published in Russian in Molekulyarnaya Biologiya, 2006, Vol. 40, No. 4, pp. 684–693. The article is published in the original.     

General RNA‐binding proteins have a function in poly(A)‐binding protein‐dependent translation

The interaction between the poly(A)‐binding protein (PABP) and eukaryotic translational initiation factor 4G (eIF4G), which brings about circularization of the mRNA, stimulates translation. General RNA‐binding proteins affect translation, but their role in mRNA circularization has not been studied before. Here, we demonstrate that the major mRNA ribonucleoprotein YB‐1 has a pivotal function in the regulation of eIF4F activity by PABP. In cell extracts, the addition of YB‐1 exacerbated the inhibition of 80S ribosome initiation complex formation by PABP depletion. Rabbit reticulocyte lysate in which PABP weakly stimulates translation is rendered PABP‐dependent after the addition of YB‐1. In this system, eIF4E binding to the cap structure is inhibited by YB‐1 and stimulated by a nonspecific RNA. Significantly, adding PABP back to the depleted lysate stimulated eIF4E binding to the cap structure more potently if this binding had been downregulated by YB‐1. Conversely, adding nonspecific RNA abrogated PABP stimulation of eIF4E binding. These data strongly suggest that competition between YB‐1 and eIF4G for mRNA binding is required for efficient stimulation of eIF4F activity by PABP.     

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.     

eIF4G functionally differs from eIFiso4G in promoting internal initiation, cap-independent translation, and translation of structured mRNAs

Eukaryotic initiation factor (eIF) 4G plays an important role in assembling the initiation complex required for ribosome binding to an mRNA. Plants, animals, and yeast each express two eIF4G homologs, which share only 30, 46, and 53% identity, respectively. We have examined the functional differences between plant eIF4G proteins, referred to as eIF4G and eIFiso4G, when present as subunits of eIF4F and eIFiso4F, respectively. The degree to which a 5'-cap stimulated translation was inversely correlated with the concentration of eIF4F or eIFiso4F and required the poly(A)-binding protein for optimal function. Although eIF4F and eIFiso4F directed translation of unstructured mRNAs, eIF4F supported translation of an mRNA containing 5'-proximal secondary structure substantially better than did eIFiso4F. Moreover, eIF4F stimulated translation from uncapped monocistronic or dicistronic mRNAs to a greater extent than did eIFiso4F. These data suggest that at least some functions of plant eIFiso4F and eIF4F have diverged in that eIFiso4F promotes translation preferentially from unstructured mRNAs, whereas eIF4F can promote translation also from mRNAs that contain a structured 5'-leader and that are uncapped or contain multiple cistrons. This ability may also enable eIF4F to promote translation from standard mRNAs under cellular conditions in which cap-dependent translation is inhibited.     

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.