Poly(A)-binding protein promotes VPg-dependent translation of potyvirus through enhanced binding of phosphorylated eIFiso4F and eIFiso4F∙eIF4B.

The phosphorylation of eukaryotic translational initiation factors has been shown to play a significant role in controlling the synthesis of protein. Viral infection, environmental stress, and growth circumstances cause phosphorylation or dephosphorylation of plant initiation factors. Our findings indicate that casein kinase 2 can phosphorylate recombinant wheat eIFiso4E and eIFiso4G generated from E. coli in vitro. For wheat eIFiso4E, Ser-207 was found to be the in vitro phosphorylation site. eIFiso4E lacks an amino acid that can be phosphorylated at the position corresponding to Ser-209, the phosphorylation site in mammalian eIF4E, yet phosphorylation of eIFiso4E has effects on VPg binding affinity that are similar to those of phosphorylation of mammalian eIF4E. The addition of VPg and phosphorylated eIFiso4F to depleted wheat germ extract (WGE) leads to enhancement of translation of both uncapped and capped viral mRNA. The addition of PABP together with eIFiso4Fp and eIF4B to depleted WGE increases both uncapped and capped mRNA translation. However, it exhibits a translational advantage specifically for uncapped mRNA, implying that the phosphorylation of eIFiso4F hinders cap binding while promoting VPg binding, thereby facilitating uncapped translation. These findings indicate TEV virus mediates VPg-dependent translation by engaging a mechanism entailing phosphorylated eIFiso4Fp and PABP. To elucidate the molecular mechanisms underlying these observed effects, we studied the impact of PABP and/or eIF4B on the binding of VPg with eIFiso4Fp. The inclusion of PABP and eIF4B with eIFiso4Fp resulted in about 2-fold increase in affinity for VPg (Kd = 24 ± 1.7 nM), as compared to the affinity of eIFiso4Fp alone (Kd = 41.0 ± 3.1 nM). The interactions between VPg and eIFiso4Fp were determined to be both enthalpically and entropically favorable, with the enthalpic contribution accounting for 76-97% of the ΔG at 25°C, indicating a substantial role of hydrogen bonding in enhancing the stability of the complex. The binding of PABP to eIFiso4Fp·4B resulted in a conformational alteration, leading to a significant enhancement in the binding affinity to VPg. These observations suggest PABP enhances the affinity between eIFiso4Fp and VPg, leading to an overall conformational change that provides a stable platform for efficient viral translation.

Iron response elements (IREs)-mRNA of Alzheimer's amyloid precursor protein binding to iron regulatory protein (IRP1): a combined molecular docking and spectroscopic approach.

The interaction between the stem-loop structure of the Alzheimer's amyloid precursor protein IRE mRNA and iron regulatory protein was examined by employing molecular docking and multi-spectroscopic techniques. A detailed molecular docking analysis of APP IRE mRNA∙IRP1 reveals that 11 residues are involved in hydrogen bonding as the main driving force for the interaction. Fluorescence binding results revealed a strong interaction between APP IRE mRNA and IRP1 with a binding affinity and an average binding sites of 31.3 × 106 M-1 and 1.0, respectively. Addition of Fe2+(anaerobic) showed a decreased (3.3-fold) binding affinity of APP mRNA∙IRP1. Further, thermodynamic parameters of APP mRNA∙IRP1 interactions were an enthalpy-driven and entropy-favored event, with a large negative ΔH (-25.7 ± 2.5 kJ/mol) and a positive ΔS (65.0 ± 3.7 J/mol·K). A negative ΔH value for the complex formation suggested the contribution of hydrogen bonds and van der Waals forces. The addition of iron increased the enthalpic contribution by 38% and decreased the entropic influence by 97%. Furthermore, the stopped-flow kinetics of APP IRE mRNA∙IRP1 also confirmed the complex formation, having the rate of association (kon) and the rate of dissociation (koff) as 341 µM-1 s-1, and 11 s-1, respectively. The addition of Fe2+ has decreased the rate of association (kon) by ~ three-fold, whereas the rate of dissociation (koff) has increased by ~ two-fold. The activation energy for APP mRNA∙IRP1 complex was 52.5 ± 2.1 kJ/mol. The addition of Fe2+ changed appreciably the activation energy for the binding of APP mRNA with IRP1. Moreover, circular dichroism spectroscopy has confirmed further the APP mRNA∙IRP1 complex formation and IRP1 secondary structure change with the addition of APP mRNA. In the interaction between APP mRNA and IRP1, iron promotes structural changes in the APP IRE mRNA∙IRP1 complexes by changing the number of hydrogen bonds and promoting a conformational change in the IRP1 structure when it is bound to the APP IRE mRNA. It further illustrates how IRE stem-loop structure influences selectively the thermodynamics and kinetics of these protein-RNA interactions.

Ferritin Iron Responsive Elements (IREs) mRNA Interacts with eIF4G and Activates In Vitro Translation.

BACKGROUND: Eukaryotic initiation factor (eIF) 4G plays an important role in assembling the initiation complex required for ribosome binding to mRNA and promote translation. Translation of ferritin IRE mRNAs is regulated by iron through iron responsive elements (IREs) and iron regulatory protein (IRP). The noncoding IRE stem-loop (30-nt) structure control synthesis of proteins in iron trafficking, cell cycling, and nervous system function. High cellular iron concentrations promote IRE RNA binding to ribosome and initiation factors, and allow synthesis of ferritin. METHODS: In vitro translation assay was performed in depleted wheat germ lysate with supplementation of initiation factors. Fluorescence spectroscopy was used to characterize eIF4F/IRE binding. RESULTS: Eukaryotic initiation factor eIF4G increases the translation of ferritin through binding to stem loop structure of iron responsive elements mRNA in the 5'-untranslated region. Our translation experiment demonstrated that exogenous addition of eIF4G selectively enhanced the translation of ferritin IRE RNA in depleted WG lysate. However, eIF4G facilitates capped IRE RNA translation significantly higher than uncapped IRE RNA translation. Addition of iron with eIF4G to depleted WG lysate significantly enhanced translation for both IRE mRNA (capped and uncapped), confirming the contribution of eIF4G and iron as a potent enhancer of ferritin IRE mRNA translation. Fluorescence data revealed that ferritin IRE strongly interacts to eIF4G (Kd = 63 nM), but not eIF4E. Further equilibrium studies showed that iron enhanced (~4-fold) the ferritin IRE binding to eIF4G. The equilibrium binding effects of iron on ferritin IRE RNA/eIFs interaction and the temperature dependence of this reaction were measured and compared. The Kd values for the IRE binding to eIF4G ranging from 18.2 nM to 63.0 nM as temperature elevated from 5 °C to 25 °C, while the presence of iron showed much stronger affinity over the same range of temperatures. Thermodynamic parameter revealed that IRE RNA binds to eIF4G with ΔH = -42.6 ± 3.3 kJ. mole-1, ΔS = -11.5 ± 0.4 J. mole-1K-1, and ΔG = -39.2 ± 2.7 kJ. mole-1, respectively. Furthermore, addition of iron significantly changed the values of thermodynamic parameters, favoring stable complex formation, thus favoring efficient protein synthesis. This study first time demonstrate the participation of eIF4G in ferritin IRE mRNA translation. CONCLUSIONS: eIF4G specifically interacts with ferritin IRE RNA and promotes eIF4G-dependent translation.

Phosphorylation of eukaryotic initiation factor eIFiso4E enhances the binding rates to VPg of turnip mosaic virus.

Binding of phosphorylated eIFiso4E with viral genome-linked protein (VPg) of turnip mosaic virus was examined by stopped-flow, fluorescence, circular dichroism (CD) spectroscopy, and molecular docking analysis. Phosphorylation of eIFiso4E increased (4-fold) the binding rates as compared to unphosphorylated eIFiso4E with VPg. Stopped-flow kinetic studies of phosphorylated eIFiso4E with VPg showed a concentration-independent conformational change. The dissociation rate was about 3-fold slower for eIFiso4E∙VPg complex upon phosphorylation. Phosphorylation enhanced the association rates and lowered the dissociation rates for the eIFiso4E∙VPg binding, with having higher preferential binding to eIFiso4Ep. Binding rates for the interaction of eIFiso4Ep with VPg increased (6-fold) with an increase in temperature, 278 K to 298 K. The activation energies for binding of eIFiso4Ep and eIFiso4E with VPg were 37.2 ± 2.8 and 52.6 ± 3.6 kJ/mol, respectively. Phosphorylation decreased the activation energy for the binding of eIFiso4E to VPg. The reduced energy barrier suggests more stable platform for eIFiso4Ep∙VPg initiation complex formation, which was further supported by molecular docking analysis. Moreover, far-UV CD studies revealed that VPg formed complex with eIFiso4Ep with substantial change in the secondary structure. These results suggested that phosphorylation, not only reduced the energy barrier and dissociation rate but also enhanced binding rate, and an overall conformational change, which provides a more stable platform for efficient viral translation.

Iron enhances the binding rates and translational efficiency of iron responsive elements (IREs) mRNA with initiation factor eIF4F.

Interaction of iron responsive elements (IRE) mRNA with the translational machinery is an early step critical in the initiation of protein synthesis. To investigate the binding specificity of IRE mRNA for eIF4F, kinetic rates for the eIF4F·IRE RNA interactions were determined and correlated with the translational efficiency. The observed rate of eIF4F·FRT IRE RNA interactions was 2-fold greater as compared to eIF4F·ACO2 IRE RNA binding. Addition of iron enhanced the association rates and lowered the dissociation rates for the eIF4F binding to both IRE RNAs, with having higher preferential binding to the FRT IRE RNA. The binding rates of both eIF4F·IRE RNA complexes correlated with the enhancement of protein synthesis in vitro. Presence of iron and eIF4F in the depleted WGE significantly enhanced translation for both IRE RNAs. This suggests that iron promotes translation by enhancing the binding rates of the eIF4F∙IRE RNA complex. eIF4F·IRE RNA binding is temperature-dependent; raising the temperature from 5 to 25°C, enhanced the binding rates of eIF4F·FRT IRE (4-fold) and eIF4F·ACO2 IRE (5-fold). Presence of Fe2+ caused reduction in the activation energy for the binding of FRT IRE and ACO2 IRE to eIF4F, suggesting a more stable platform for initiating protein synthesis. In the presence of iron, lowered energy barrier has leads to the faster association rate and slower rate of dissociation for the protein-RNA complex, thus favoring efficient protein synthesis. Our results correlate well with the observed translational efficiency of IRE RNA, thereby suggesting that the presence of iron leads to a rapid, favorable, and stable complex formation that directs regulatory system to respond efficiently to cellular iron levels.

Interaction of ferritin iron responsive element (IRE) mRNA with translation initiation factor eIF4F.

The interaction of ferritin iron responsive element (IRE) mRNA with eIF4F was examined by fluorescence and circular dichroism spectroscopy. Fluorescence quenching data indicated that eIF4F contains one high affinity binding site for ferritin IRE RNA. The Scatchard analysis revealed strong binding affinity (Ka = 11.1 × 107 M-1) and binding capacity (n = 1.0) between IRE RNA and eIF4F. The binding affinity of IRE RNA for eIF4F decreased (~4-fold) as temperature increased (from 5 °C to 30 °C). The van't Hoff analysis revealed that IRE RNA binding to eIF4F is enthalpy-driven (ΔH = -47.1 ± 3.4 kJ/mol) and entropy-opposed (ΔS = -30.1 ± 1.5 J/mol/K). The addition of iron increased the enthalpic, while decreasing the entropic contribution towards the eIF4F•IRE RNA complex, resulting in favorable free energy (ΔG = -49.8 ± 2.8 kJ/mol). Thermodynamic values and ionic strength data suggest that the presence of iron increases hydrogen bonding and decreases hydrophobic interactions, leading to formation of a more stable complex. The interaction of IRE RNA with eIF4F at higher concentrations produced significant changes in the secondary structure of the protein, as revealed from the far-UV CD results, clearly illustrating the structural alterations resulted from formation of the eIF4F•IRE RNA complex. A Lineweaver-Burk plot showed an uncompetitive binding behavior between IRE RNA and m7G cap for the eIF4F, indicating that there are different binding sites on the eIF4F for the IRE RNA and the cap analog; molecular docking analysis further supports this notion. Our findings suggest that the eIF4F•IRE RNA complex formation is accompanied by an elevated hydrogen bonding and weakened hydrophobic interactions, leading to an overall conformational change, favored in terms of its free energy. The conformational change in the eIF4F structure, caused by the IRE RNA binding, provides a more stable platform for effective IRE translation in iron homeostasis.

Phosphorylation of translation initiation factor eIFiso4E promotes translation through enhanced binding to potyvirus VPg.

Interactions of phosphorylated eIFiso4E binding to VPg as a function of temperature and ionic strength were assessed employing fluorescence spectroscopic. Phosphorylation increased the binding affinity ∼3.5-fold between VPg and eIFiso4E under equilibrium conditions. Binding affinity of VPg for eIFiso4Ep correlates with the ability to enhance in vitro protein synthesis. Addition of VPg and eIFiso4Ep together to Dep WGE enhances the translation for both uncapped and capped mRNA. However, capped mRNA translation was inhibited with addition of eIFiso4Ep alone in dep WGE, suggesting that phosphorylation prevents the cap binding and favours the VPg binding to promotes translation. Temperature dependence showed that the phosphorylated form of the eIFiso4E is preferred for complex formation. A van't Hoff analysis reveals that eIFiso4Ep binding to VPg was enthalpy driven (ΔH = -43.9 ± 0.3 kJ.mol-1) and entropy-opposed (ΔS = -4.3 ± 0.1 J.mol-1K-1). Phosphorylation increased the enthalpic contributions ∼33% for eIFiso4Ep-VPg complex. The thermodynamic values and ionic strength dependence of binding data suggesting that phosphorylation increased hydrogen-bonding and decreased hydrophobic interactions, which leads to more stable complex formation and favour efficient viral translation. Overall these data correlate well with the observed translational data and provide more detailed information on the translational strategy of potyviruses.

Poly (A) binding protein enhances the binding affinity of potyvirus VPg to eukaryotic initiation factor eIF4F and activates in vitro translation.

Viral protein linked to the genome (VPg) of turnip mosaic virus (TuMV) interacts with eIF4F and plays an important role in cap-independent initiation of protein synthesis. In order to understand the importance of PABP on the interaction of eIF4F with VPg, we report that PABP enhanced the binding affinity ~3- and 4-fold for eIF4F-VPg and eIF4F·eIF4B-VPg, respectively. PABP enhances rates of protein synthesis in uncapped viral mRNA and correlates with binding affinity of eIF4F with VPg. Temperature dependent (278 K to 298 K) showed ~3-fold increase in eIF4F binding to VPg in presence of PABP and eIF4B. A van't Hoff analysis reveals that eIF4F·eIF4B·PABP binding to VPg was enthalpy-driven and entropy-favorable with 30% increase in enthalpic contribution and 81% decrease in entropic contribution. PABP increased the association rate (4-fold) and decreased the dissociation rate (3-fold) for the eIF4F·eIF4B binding to VPg. PABP significantly decreased the activation energy of eIF4F·eIF4B binding to VPg. When both PABP and eIF4B were present, not only was the energy barrier reduced but the binding rate was faster and dissociation rate was slower for the protein-protein complex formation. These results suggest increased hydrogen bonding and an overall conformational change, and more stable platform for effective viral translation.

Kinetic analyses of phosphorylated and non-phosphorylated eIFiso4E binding to mRNA cap analogues.

Phosphorylation of eukaryotic initiation factors was previously shown to interact with m7G cap and play an important role in the regulation of translation initiation of protein synthesis. To gain further insight into the phosphorylation process of plant protein synthesis, the kinetics of phosphorylated wheat eIFiso4E binding to m7G cap analogues were examined. Phosphorylation of wheat eIFiso4E showed similar kinetic effects to human eIF4E binding to m7-G cap. Phosphorylation of eIFiso4E decreased the kinetic rate (2-fold) and increased the dissociation rate (2-fold) as compared to non-phosphorylated eIFiso4E binding to both mono- and di-nucleotide analogues at 22°C. Phosphorylated and non-phosphorylated eIFiso4E-m7G cap binding rates were found to be independent of concentration, suggesting conformational changes were rate limiting. Rate constant for phosphorylated and non-phosphorylated eIFiso4E binding to m7-G cap increased with temperature. Phosphorylation of eIFiso4E decreased (2-fold) the activation energy for both m7-G cap analogues binding as compared to non-phosphorylated eIFiso4E. The reduced energy barrier for the formation of eIFiso4E-m7-G cap complex suggests a more stable platform for further initiation complex formation and possible means of adapting variety of environmental conditions. Furthermore, the formation of phosphorylated eIFiso4E-cap complex may contribute to modulation of the initiation of protein synthesis in plants.

Thermodynamic and Kinetic Analyses of Iron Response Element (IRE)-mRNA Binding to Iron Regulatory Protein, IRP1.

Comparison of kinetic and thermodynamic properties of IRP1 (iron regulatory protein1) binding to FRT (ferritin) and ACO2 (aconitase2) IRE-RNAs, with or without Mn2+, revealed differences specific to each IRE-RNA. Conserved among animal mRNAs, IRE-RNA structures are noncoding and bind Fe2+ to regulate biosynthesis rates of the encoded, iron homeostatic proteins. IRP1 protein binds IRE-RNA, inhibiting mRNA activity; Fe2+ decreases IRE-mRNA/IRP1 binding, increasing encoded protein synthesis. Here, we observed heat, 5 °C to 30 °C, increased IRP1 binding to IRE-RNA 4-fold (FRT IRE-RNA) or 3-fold (ACO2 IRE-RNA), which was enthalpy driven and entropy favorable. Mn2+ (50 µM, 25 °C) increased IRE-RNA/IRP1 binding (K d) 12-fold (FRT IRE-RNA) or 6-fold (ACO2 IRE-RNA); enthalpic contributions decreased ~61% (FRT) or ~32% (ACO2), and entropic contributions increased ~39% (FRT) or ~68% (ACO2). IRE-RNA/IRP1 binding changed activation energies: FRT IRE-RNA 47.0 ± 2.5 kJ/mol, ACO2 IRE-RNA 35.0 ± 2.0 kJ/mol. Mn2+ (50 µM) decreased the activation energy of RNA-IRP1 binding for both IRE-RNAs. The observations suggest decreased RNA hydrogen bonding and changed RNA conformation upon IRP1 binding and illustrate how small, conserved, sequence differences among IRE-mRNAs selectively influence thermodynamic and kinetic selectivity of the protein/RNA interactions.

Rapid kinetics of iron responsive element (IRE) RNA/iron regulatory protein 1 and IRE-RNA/eIF4F complexes respond differently to metal ions.

Metal ion binding was previously shown to destabilize IRE-RNA/IRP1 equilibria and enhanced IRE-RNA/eIF4F equilibria. In order to understand the relative importance of kinetics and stability, we now report rapid rates of protein/RNA complex assembly and dissociation for two IRE-RNAs with IRP1, and quantitatively different metal ion response kinetics that coincide with the different iron responses in vivo. kon, for FRT IRE-RNA binding to IRP1 was eight times faster than ACO2 IRE-RNA. Mn(2+) decreased kon and increased koff for IRP1 binding to both FRT and ACO2 IRE-RNA, with a larger effect for FRT IRE-RNA. In order to further understand IRE-mRNA regulation in terms of kinetics and stability, eIF4F kinetics with FRT IRE-RNA were determined. kon for eIF4F binding to FRT IRE-RNA in the absence of metal ions was 5-times slower than the IRP1 binding to FRT IRE-RNA. Mn(2+) increased the association rate for eIF4F binding to FRT IRE-RNA, so that at 50 µM Mn(2+) eIF4F bound more than 3-times faster than IRP1. IRP1/IRE-RNA complex has a much shorter life-time than the eIF4F/IRE-RNA complex, which suggests that both rate of assembly and stability of the complexes are important, and that allows this regulatory system to respond rapidly to change in cellular iron.

Fe2+ binds iron responsive element-RNA, selectively changing protein-binding affinities and regulating mRNA repression and activation.

Iron increases synthesis rates of proteins encoded in iron-responsive element (IRE)-mRNAs; metabolic iron ("free," "labile") is Fe(2+). The noncoding IRE-RNA structure, approximately 30 nt, folds into a stem loop to control synthesis of proteins in iron trafficking, cell cycling, and nervous system function. IRE-RNA riboregulators bind specifically to iron-regulatory proteins (IRP) proteins, inhibiting ribosome binding. Deletion of the IRE-RNA from an mRNA decreases both IRP binding and IRP-independent protein synthesis, indicating effects of other "factors." Current models of IRE-mRNA regulation, emphasizing iron-dependent degradation/modification of IRP, lack answers about how iron increases IRE-RNA/IRP protein dissociation or how IRE-RNA, after IRP dissociation, influences protein synthesis rates. However, we observed Fe(2+) (anaerobic) or Mn(2+) selectively increase the IRE-RNA/IRP K(D). Here we show: (i) Fe(2+) binds to the IRE-RNA, altering its conformation (by 2-aminopurine fluorescence and ethidium bromide displacement); (ii) metal ions increase translation of IRE-mRNA in vitro; (iii) eukaryotic initiation factor (eIF)4F binds specifically with high affinity to IRE-RNA; (iv) Fe(2+) increased eIF4F/IRE-RNA binding, which outcompetes IRP binding; (v) exogenous eIF4F rescued metal-dependent IRE-RNA translation in eIF4F-depeleted extracts. The regulation by metabolic iron binding to IRE-RNA to decrease inhibitor protein (IRP) binding and increase activator protein (eIF4F) binding identifies IRE-RNA as a riboregulator.

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.

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 analysis of the interaction of b/HLH/Z transcription factors Myc, Max, and Mad with cognate DNA.

Myc, Mad, and Max proteins belong to the basic helix-loop-helix leucine zipper family of transcription factors. They bind to a specific hexanucleotide element of DNA, the E-box (CACGTG). To be biologically active, Myc and Mad require dimerization with Max. For the route of complex assembly of these dimers, there are two proposed pathways. In the monomer pathway, two monomers bind DNA sequentially and assemble their dimerization interface while bound to DNA. In the dimer pathway, two monomers form a dimer first prior to association with DNA. The monomer pathway is kinetically favored. In this report, stopped-flow polarization was utilized to determine the rates and temperature dependence of all of the individual steps for both assembly pathways. Myc.Max dimerization had a rate constant approximately 5- and approximately 2-fold higher than those of Max.Max and Mad.Max dimerization, respectively. The protein dimerization rates as well as the dimer-DNA rates were found to be independent of concentration, suggesting conformational changes were rate-limiting. The Arrhenius activation energies for the dimerization of Myc, Mad, and Max with Max were 20.4 +/- 0.8, 29 +/- 0.6, and 40 +/- 0.2 kJ/mol, respectively. Further, rate constants for Max.Max homodimer DNA binding are significantly higher than for Myc.Max and Mad.Max heterodimers binding to DNA. Monomer-DNA binding showed a faster rate than dimer-DNA binding. These studies show the rate-limiting step for the dimer pathway is the formation of protein dimers, and this reaction is slower than formation of protein dimers on the DNA interface, kinetically favoring the monomer pathway.

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 micro 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 approximately 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.

Direct Fe2+ sensing by iron-responsive messenger RNA:repressor complexes weakens binding.

Fe(2+) is now shown to weaken binding between ferritin and mitochondrial aconitase messenger RNA noncoding regulatory structures ((iron-responsive element) (IRE)-RNAs) and the regulatory proteins (IRPs), which adds a direct role of iron to regulation that can complement the well known regulatory protein modification and degradative pathways related to iron-induced mRNA translation. We observe that the K(d) value increases 17-fold in 5'-untranslated region IRE-RNA:repressor complexes; Fe(2+), is studied in the absence of O(2). Other metal ions, Mn(2+) and Mg(2+) have similar effects to Fe(2+) but the required Mg(2+) concentration is 100 times greater than for Fe(2+) or Mn(2+). Metal ions also weaken ethidium bromide binding to IRE-RNA with no effect on IRP fluorescence, using Mn(2+) as an O(2)-resistant surrogate for Fe(2+), indicating that metal ions bound IRE-RNA but not IRP: Fe(2+) decreases IRP repressor complex stability of ferritin IRE-RNA 5-10 times compared with 2-5 times for mitochondrial aconitase IRE-RNA, over the same concentration range, suggesting that differences among IRE-RNA structures contribute to the differences in the iron responses observed in vivo. The results show the IRE-RNA:repressor complex literally responds to Fe(2+), selectively for each IRE-mRNA.

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.

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.

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.