30-nucleotide (sequence: 5${}^{\prime }$-GUUCUUGCUUCAACAGUGUUUGAACGGAAC-3${}^{\prime }$) ferritin IRE mRNA and 5S RNA 30-nt (5${}^{\prime }$-UAGUACUUGGAUGGGAGACCGCCUGGGA AU-3${}^{\prime }$) were purchase from Metabion. Preparation of RNAs were performed as described in references [21, 24]. The concentrations of ferritin IRE RNA and 5S RNA solutions were determined by measuring the optical density at $\lambda$${}_{\text{260 nm}}$ spectrophotometrically. Recombinant protein of human eukaryotic translation initiation factors eIF4G1 (Catalog-TP312877, Lot-107AB8) and eIF4E (Catalog-TP721168, Lot-0330637) were supplied by OriGene Co. (Rockville, MD). Proteins expressed and purified from mammalian cells using HeK293T cells, a line derived from human embryonic kidney. Proteins are purified using an anti-DDK immunoaffinity column followed by conventional chromatography steps as described in OriGene protocols. Human eIF4E and eIF4G1 stock solutions were prepared by dissolving fix amount of protein crystals in 1 mL of 20 mM HEPES/KOH buffer, pH 7.2, and its concentrations were measured spectrophotometrically using Bradford method. Ferritin mRNA construct was transcribed and purified as described previously [25]. Capped mRNA was synthesized by using the Ribo m${}^7$G cap analog reaction protocol provided by Promega. Under optimized conditions, above 95% RNA was capped. Wheat germ lysate was supplied by Promega. Wheat germ lysate contain all the cellular components necessary for protein synthesis.

Ferritin mRNA transcript (capped and uncapped) were translated in wheat germ (WG) lysate and depleted wheat germ (WG) lysate as described previously [4, 26]. Translation assay of depleted WG lysate is dependent on the supplementation of eIF4G and eIF4E. eIF4F depleted WG lysate were prepared following Promega instructions manual. WG lysate (200 $\mu$L) was mixed to m${}^7$GTP-Sepharose (300 $\mu$L) and incubated with continuous rotation for half an hour at 5 °C. The column was washed with standard buffer: 20 mM HEPES/KOH (pH 7.2), and eukaryotic initiation factors eluted with above buffer containing 100 mM GTP. The extent of depletion of the eIF4G and eIF4E from wheat germ lysate was determined by Western blot analysis following resolution of the extract by SDS-PAGE as shown previously [27]. Western blot analysis confirmed that the level of eIF4G and eIF4E was reduced by about 95%. Capped and uncapped transcript mRNA (IRE luciferase reporter mRNA) was assayed with WG lysate and depleted WG lysate according to the manufacture instructions. For all reaction mixture transcript RNA (capped and uncapped) was heated at 85 °C for 15 min, and the reaction mixture annealed slowly at room temperature for 30 min. When iron was added, all sample incubations were anaerobic. Anaerobiosis conditions for iron was achieved with using argon in a glass sealed vials as previously described [21]. Briefly, 50 (l reaction mixture included 10 $\mu$g ferritin IRE-luc mRNA template; 25 $\mu$L of either WG lysate or depleted WG lysate; 1 mM amino acids (set of 19 amino acids); 40 units of RNasin Ribonuclease, 100 mM potassium chloride; 2 mM magnesium chloride [28, 29]. Incubation of titration mixture was for 120 min at 25 °C. depleted WG lysate translation is dependent upon addition of eIF4G (100 nM) and eIF4E (100 nM). To investigate the eIF4G-dependent translation, depleted WG lysate was added with 100 nM eIF4G or eIF4E in translation reaction mixture. Translation was also assayed with addition of Fe${}^{2+}$ (50 micro M). When Fe${}^{2+}$ was used, experiments were anaerobic conditions. Nitrogen-purged solution of 0.1 M HCl used to dissolve FeSO${}_4$ as described previously [21]. After addition of the luciferase assay reagent (100 $\mu$L) to each translation sample, the amount of protein expression was determined by measuring the light produced by Luminometer at 495 nm. Control sample containing no RNA, were used to measurement of any background absorbance.

The titration experiments for the ferritin IRE RNA interaction with eIF4G, eIF4E or eIF4G$\cdot$eIF4E complex were performed by fluorescence intensity measurements as previously described [30]. Protein fluorescence was excited at $\lambda$${}_{\text{Ex}}$ 280 nm, and the data was collected at $\lambda$${}_{\text{Em}}$ of 340 nm. Titration experiments were performed for 0.1 $\mu$M eIF4G, eIF4E, or eIF4G$\cdot$eIF4E complex with addition of different amount ferritin IRE in steady-state conditions provided by pre-incubation in standard buffer 20 mM HEPES/KOH (pH 7.2), at ionic strength of 100 mM KCl, 2 mM MgCl${}_2$, and 5% (v/v) glycerol. The binding of eIF4G and eIF4E with ferritin IRE RNA were measured by quenching of intrinsic fluorescence of protein. The reaction mixture was thermostat, and the cuvette temperature was maintained ($\mathrm{\Delta }$T $\pm$ 0.2 °C) for all temperature-dependent binding experiments. Before the fluorescence measurements, the samples were incubated for 20 min at appropriate temperature. Binding of eIF4G, eIF4E, or eIF4G$\cdot$eIF4E with ferritin IRE RNA were determined by measuring the intrinsic fluorescence intensity of eIFs protein and protein-IRE RNA solution. Normalized fluorescence ($\mathrm{\Delta }$F/$\mathrm{\Delta }$F${}_{\text{max}}$) between eIFs-IRE complex and individual eIFs intrinsic fluorescence intensity were used to measure the dissociation constant (K${}_{\mathrm{\text{d}}}$) as described previously [30, 31]. In control experiments, fluorescence intensity of eIFs (0.1 $\mu$M) alone was measured. In another sample fluorescence intensity of ferritin IRE RNA at specific concentration was measured. Control fluorescence intensity was used to determine the corrected fluorescence intensity of the complex. The fluorescence data obtained above were corrected for dilution and blank contributions, if any, as described previously [27]. All solutions were filtered prior to the measurements. The fluorescence spectra are the average of three individual spectrum. The fluorescence data were fit by means of nonlinear least squares method, using Kaleida-Graph Software (Version 2.1.3; Synergy Abelbeck Software Inc. USA)

The number of IRE binding site on the eukaryotic initiation factor eIF4G were measured by exciting the protein and protein-RNA complex at $\lambda$${}_{\text{max}}$ 280 nm and fluorescence intensity was recorded at emission $\lambda$${}_{\text{max}}$ 340 nm. Protein (eIF4G) fluorescence quenching was measured with addition of varying amount of ferritin IRE RNA. For each molar ratio of RNA/eIF4G, relative fluorescence change compared to control (untreated eIF4G fluorescence) were recorded. The fluorescence intensity of protein (F) as a function of ferritin IRE RNA experimental data was fitted according to the following equation: Q = (F${}_0$ – F)/m. where Q and m are the fractional and maximal quench of eIF4G protein with and without addition of RNA. Fractional quench (Q) of eIF4G is directly related with RNA binding following equation Q = [eIF4G-IRE]/[eIF4G]${}_{\text{T}}$, where [eIF4G]${}_{\text{T}}$ is the total amount of protein in solution. Q is related to the equilibrium concentration of the ratio of eIF4G/IRE complex and eIF4G total. The resulting data were expressed as number of binding sites for IRE on eIF4G as described previously using the Scatchard analysis [30, 32, 33]. K${}_{\mathrm{\text{a}}}$ values were obtained from the Scatchard plot and nonlinear least square fitting were similar. The fluorescence data were fit to nonlinear least square using Kaleida Graph software (Version 2.1.3; Synergy Abelbeck Software Inc. USA).

For competitive binding measurements, the molar ratio of eIF4G to eIF4E was 1:0, 1:1, and 1:2. eIF4G concentration was kept constant at 0.1 $\mu$M. Titration were performed at several eIF4E concentrations (0.0, 100, and 200 nM) as a function of IRE in steady state conditions, provided by pre-incubation of titration samples for 15 min. Titration experiments were performed in standard buffer (20 mM HEPES/KOH, pH 7.2) containing 100 mM KCl and 2 mM MgCl${}_2$. Fluorescence change of eukaryotic initiation factor eIF4G with increasing concentrations of IRE-mRNA were recorded by spectroscopic measurements. For eIF4G/IRE binding measurements the excitation and emission $\lambda$${}_{\text{max}}$ 280 nm (slit width 4 nm) and 332 nm (slit width 5 nm) were used by observing the fluorescence signal change of the eIF4G protein and protein/RNA complex solution. The data were fit by non-linear least-square using Kaleida Graph software. The characteristic of competitive and uncompetitive binding was determined from the Lineweaver-Burk plot as described previously [4, 30].

Temperature dependence of dissociation constant were used to construct van’t Hoff plots according to the equation:

(1)$$\ln K_{\mathrm{a}}\mathit{\hspace{1em}}=\mathit{\hspace{1em}}-[\mathrm{\Delta }\mathrm{H}]/[\mathrm{RT}]+\mathit{\hspace{1em}}+\mathit{\hspace{1em}}[\mathrm{\Delta }\mathrm{S}]/[\mathrm{R}]$$

where K${}_{\mathrm{\text{a}}}$ is the binding affinity at each temperature (5, 10, 15, 20, and 25 °C). The entropy changes $\mathrm{\Delta }$S and enthalpy change $\mathrm{\Delta }$H were calculated using the intercept and slope of lnK${}_{\mathrm{\text{a}}}$ versus temperature (T${}^{-1}$). The change in free energy $\mathrm{\Delta }$G of the binding reaction was calculated at 25 °C by the following equation:

(2)$$\mathrm{\Delta }\mathrm{G}=\mathrm{\Delta }\mathrm{H}-\mathrm{T}\mathrm{\Delta }\mathrm{S}\mathit{\hspace{1em}}\text{and}\mathit{\hspace{1em}}\mathrm{\Delta }\mathrm{G}=-\mathrm{RT}\ln K_{\mathrm{a}}$$

We have previously [4] shown that ferritin IRE interacts with eIF4F, but functional differences between two subunits of eIF4F, eIF4G and eIF4E in protein synthesis and specificity binding has not been elucidated. We have correlated the binding affinity with translation efficiency of individual subunits. To determine whether the two subunits eIF4G and eIF4E were able to support capped or uncapped ferritin luciferase mRNA translation in eIF4F-dependent WG lysate was prepared [34]. eIF4F-depleted lysate was generated by binding of WG extract to m${}^7$GTP-Sepharose. Western blot analysis confirmed that the level of eIF4G and eIF4E was reduced by 95%. To assess the capped/uncapped ferritin mRNA translation, we used a complete WG lysate and depleted WG lysate that was supplemented with the subunits eIF4G and/or eIF4E initiation factors. Depletion of eIF4F from the wheat germ lysate reduced translation by more than 95% (compare WG lysate and depleted WG lysate translation) (Figs. 1,2). Adding capped or uncapped ferritin mRNA to a complete wheat germ lysate significantly enhanced protein synthesis. However, addition of capped or uncapped ferritin IRE RNA to depleted lysate of wheat germ did not support translation in vitro (Fig. 1). Supplementation of eIF4G to depleted wheat germ lysate enhanced translation of capped and uncapped ferritin mRNA up to 51- and 40-fold (Fig. 1), whereas supplementation of eIF4E to the lysate of DWG did not appear translational difference of capped and uncapped ferritin IRE mRNA. As shown in Fig. 1, the addition of both subunits eIF4G and eIF4E (mixed complex) can fully support translation of ferritin mRNA. Interestingly, translation efficiency appears to correlate more closely with eIF4G than eIF4E subunit. Furthermore, wheat germ translation was largely dependent on eIF4G. Translation results suggest that capped/uncapped mRNA provides eIF4G-dependent translation, whereas capped mRNA facilitates high level of translation as compared to uncapped mRNA through eIF4G binding. Moreover, Addition of iron and eIF4G to depleted wheat germ lysate increases translation ~2-fold for both ferritin mRNA.

Fig. 1.

eIF4G enhances capped ferritin IRE translation in eIF4F-depleted wheat germ extract. Translation assay of WG lysate and depleted WG lysate contain 10 g mRNA (capped). Depleted WG lysate was supplemented with 100 nM eIF4G or eIF4E. Concentration of Fe${}^{2+}$ was 50 $\mu$M. Point represents the average of three experiments, and the error bars are indicated.

Fig. 2.

Translation assay of uncapped ferritin IRE with eIF4G. Uncapped ferritin mRNA (10 g) was assayed in WG lysate and depleted WG lysate. Depleted WG lysate was supplemented with eIF4G (100 nM) and Fe${}^{2+}$ 50 (M), respectively. Each uncapped mRNA represents the average of three experiments, and the error bars are reported.

To examine the effect of inhibitor (IRP1), on the translation of capped/uncapped ferritin IRE RNA, eIF4G, eIF4E-depleted lysate was program to determine the degree to which translation was inhibited. The results showing (Fig. 3) that IRP1 inhibits translation of ferritin IRE RNA both in complete WG lysate and depleted WG lysate supplemented with eIF4G. Addition of 100 nM IRP1 inhibited 40–45% in vitro translation from capped/uncapped mRNA was observed in complete WG lysate, whereas 40–50% inhibition was observed in depleted WG lysate supplemented with eIF4G. These data suggest that IRP1 preferentially binds to ferritin IRE RNA as a result translation inhibited. Further, addition of 50 (M Fe${}^{2+}$ in DWG lysate supplemented with eIF4G restored translation by 90% for capped/uncapped mRNA by stabilizing eIF/IRE RNA complex [4] and destabilizing IRP1/IRE RNA complex to promote translation [21]. Iron reversed IRP1 inhibition of protein synthesis (Fig. 3A and B) by inducing release of IRP1 from IRE RNA and promote eIFs binding. Increasing cellular iron concentrations will lower IRP1/IRE RNA affinity and higher eIF4G binding, ribosome assembly, and ferritin mRNA translation.

Fig. 3.

Inhibition in translation of ferritin IRE mRNA in WG lysate and eIF4G-dependent lysate by IRP1. (A) Capped IRE RNA and (B) uncapped IRE RNA was translated in the WG lysate and depleted WG lysate. IRP1 concentration was 100 nM. Other conditions are shown in Fig. 1.

To assess the initiation factor concentration on the translation, we performed translation experiments with different amount of eIF4G or eIF4E with both capped and uncapped ferritin IRE-RNA in depleted WG lysate. As the concentration of eIF4G was increased translation of ferritin mRNA was enhanced irrespective of capped/uncapped IRE RNA (Fig. 4). Supplementation of 50 nM eIF4G enhanced 58-fold of translation for the capped IRE RNA (Fig. 4) and 54-folds of translation for uncapped IRE RNA in WG lysate. In contrast, supplementation of eIF4E did not produce any significant change on translation level of either capped or uncapped ferritin IRE RNA. As eIF4E cannot bind to ferritin IRE RNA and eIF4G strongly interacts with IRE RNA, these data appear to correlate more closely with translational efficiency with eIF4G rather than the cap binding to eIF4E. We observed that eIF4G increased cap-dependent translation efficiency to a higher level than the uncapped IRE mRNA translation. Supplementation of exogenous eIF4G initiation factor to the depleted wheat germ lysate resulted in 85% recovery of in vitro translation of ferritin mRNA, confirming that eIF4G specifically recognizes the ferritin mRNA. Furthermore, addition of iron increases the eIF4G$\cdot$IRE RNA binding affinity as illustrated by enhanced protein synthesis, resulted from iron being added to the wheat germ lysate along with exogenous eIF4G. These results strongly suggest that iron and eIF4G plays a key part in enhancing protein synthesis of ferritin regulatory elements mRNA.

Fig. 4.

eIF4G (but not eIF4E) supports in vitro translation (capped and uncapped) ferritin mRNA. Translation reaction contain eIF4F depleted wheat germ lysate with 10 (g) ferritin mRNA (capped or uncapped), and the indicated amount of recombinant eIF4G and eIF4E. Translation of capped (—$\bullet$—) and uncapped (—$\circ$—) ferritin mRNA in depleted wheat germ extract supplemented with eIF4G; for capped (—$\mathrm{▲}$—) and uncapped (—$\mathrm{△}$—) ferritin mRNA translation in depleted wheat germ extract supplemented with eIF4E.

The binding affinity of ferritin IRE RNA with large subunit eIF4G was measured to assess the specificity of complex formation. As shown in Fig. 5, eIF4G fluorescence was presented with increasing amounts of IRE RNA. Fluorescence data indicated that about 80% quenching of eIF4G fluorescence intensity with addition of ferritin IRE at the highest molar ratios. The amount of eIF4G fluorescence change is proportional to concentration of ferritin IRE-RNA binding. Fig. 5 inset indicated corresponding Scatchard plot for the eIF4G/IRE binding. Binding affinity (K${}_{\mathrm{\text{a}}}$) and number of binding site (n) of IRE RNA to eIF4G was measured by the slope and intercept of a Scatchard plot (Q/IRE] $\times$ 10${}^{-6}$ vs Q). The K${}_{\mathrm{\text{a}}}$ and n for the interaction of ferritin IRE to eIF4G were 14.8 $\times$ 10${}^6$ M${}^{-1}$ and 1.0. These results suggest that eIF4G contain one binding domain for IRE RNA. K${}_{\mathrm{\text{a}}}$ values of ferritin IRE with eIF4G were calculated by non-linear least squares method (K${}_{\mathrm{\text{d}}}$ = 63.0 nM) and Scatchard plot. Equilibrium values are reported in K${}_{\mathrm{\text{d}}}$ and K${}_{\mathrm{\text{a}}}$, whereas K${}_{\mathrm{\text{a}}}$ = 1/K${}_{\mathrm{\text{d}}}$. The results obtained by two-independent data analysis methods are in good agreement.

Fig. 5.

Titration isotherm for the binding of eIF4G with varying ferritin IRE RNA. Binding isotherm of eIF4G (100 nM) was carried out in the presence of varying ferritin IRE concentrations (at 25 °C, ${}_{\text{ex}}$ = 280 nm, ${}_{\text{em}}$ = 332 nm). The inset showing the Scatchard plot of titration data for the interaction of IRE binding with eIF4G.

To assess the ability of IRE RNA binding to the subunits eIF4G and eIF4E, we determined the binding affinity of ferritin IRE with eIF4G, eIF4E, and eIF4G$\cdot$eIF4E. eIF4G binds the ferritin IRE RNA with the dissociation constant (K${}_{\mathrm{\text{d}}}$) of 63 $\pm$ 4.3 nM (Fig. 6), similar to the binding of beta-globin mRNA [35] and several fold higher K${}_{\mathrm{\text{d}}}$ than ferritin IRE RNA/eIF4F binding [4]. 5S RNA was used as a control under the same conditions to test for non-specific binding. Conversely, no binding of eIF4G with 30-oligonucleotide stem loop from yeast 5S RNA (a negative control) was detected (Fig. 6), suggesting that ferritin IRE-RNA specifically binds to subunit eIF4G, as reported earlier for the binding of eIF4G with stem-loop structure from encephalomyocarditis viral RNA [35]. Strong binding of IRE-RNA with eIF4G, yet without the m${}^7$G cap, explains the earlier examination that removing m${}^7$G-cap from ferritin IRE does not affect the translation compared to the larger change on removing the IRE stem-loop structure [36]. The addition of eIF4E produced no significant difference on the interaction of ferritin IRE-RNA with eIF4G. However, eIF4E alone did not interact with ferritin IRE-RNA (Fig. 6). These results suggest that ferritin IRE RNA preferably binds to eIF4G, but not eIF4E. Moreover, equilibrium studies showed that iron enhanced ferritin IRE-RNA binding to eIF4G about 4-fold (eIF4G$\cdot$IRE RNA-Fe${}^{2+}$, K${}_{\text{𝑑}}$ = 17.0 nM; eIF4G$\cdot$IRE RNA, K${}_{\mathrm{\text{d}}}$ = 63 nM) at 298K. On the other hand, addition of iron did not affect the binding affinity of eIF4E to ferritin IRE (Fig. 7). As eIF4E cannot bind to ferritin mRNA, while eIF4G strongly bind to ferritin mRNA, these results suggests that eIF4G enhances in vitro translation with depleted WG lysate by binding to ferritin mRNA.

Fig. 6.

Ferritin IRE RNA tightly binds to eIF4G. The normalized fluorescence values for the binding of eIF4G (—$\circ$—), eIF4G$\cdot$eIF4E (—$\mathrm{△}$—), and eIF4E (—$\mathrm{◆}$—) versus concentration of ferritin IRE RNA. eIF4G (—$\mathrm{▲}$—) did not bind a 5S RNA 30-oligonucleotide stem loop used as a negative control. eIF4G, eIF4E, and eIF4G$\cdot$eIF4E concentration were 100 nM.

Fig. 7.

Iron enhances the ferritin IRE binding to eIF4G. Histogram representation of affinity constant (K${}_{\mathrm{\text{a}}}$$\mu$M${}^{-1}$) of ferritin IRE with eIF4E, eIF4G, and eIF4G$\cdot$eIF4E with and without iron.

To identify whether ferritin IRE RNA and eIF4E binding to eIF4G is competitive or uncompetitive, varying amount of ferritin mRNA and eIF4E were utilized. EIF4G protein fluorescence signal change was measured in the absence and presence of eIF4E with varying concentration of ferritin IRE-RNA. For ferritin IRE binding experiments, fluorescence titration results of the molar ratio of eIF4G:eIF4E was 1:0, 1:1, and 1:2, respectively. Changes in fluorescence intensity of eIF4G$\cdot$eIF4E complex at different ferritin IRE-RNA concentrations were determined. As shown in Fig. 8, Lineweaver-Burk plots show that the binding of ferritin RNA and eIF4E to eIF4G is uncompetitive. However, IRP1 and eIF4F binds to ferritin IRE RNA competitively [4]. Fluorescence results revealed that ferritin IRE and eIF4E have different binding site on eIF4G.

Fig. 8.

Ferritin IRE and eIF4E binds noncompetitively to eIF4G. Change in fluorescence intensity of eIF4G (100 nM) as a function of ferritin IRE with addition of 0.0 nM eIF4E (—$\bullet$—), 50 nM eIF4E (—$\mathrm{△}$—), and 100 nM eIF4E (—$\mathrm{▲}$—). Parallel lines of Lineweaver-Burk plots indicate uncompetitive.

The effect of temperature on eIF4G binding to ferritin IRE was observed by measuring the fluorescence intensity of protein and protein-RNA complex. The dissociation constant for eIF4G/IRE and eIF4G$\cdot$eIF4E/IRE complexes with and without addition of iron was determine as a function of temperature by observing protein fluorescence quenching data. Fluorescence results at different temperatures revealed that the binding association of eIF4G/IRE and eIF4G$\cdot$eIF4E/IRE complex enhanced with increased temperature. K${}_{\mathrm{\text{d}}}$ values increased from 18.2 $\pm$ 1.1 nM to 63 $\pm$ 4.3 nM and 9.1 $\pm$ 0.3 nM to 46.3 $\pm$ 3.6 nM for the binding of eIF4G and eIF4G$\cdot$eIF4E with IRE-RNA as temperature elevated from 5 °C to 25 °C (Table 1). Fluorescence result revealed that dissociation of eIF4G/IRE and eIF4G$\cdot$eIF4E/IRE at 5 °C were higher as compared to 25 °C (Table 1). Addition of iron (50 $\mu$M Fe${}^{2+}$) shows that eIF4G and eIF4G$\cdot$eIF4E binding to ferritin IRE increased by about 4-time. Dissociation constant for the eIF4G/IRE and eIF4G(eIF4E/IRE with addition of Fe${}^{2+}$ increased to 17.0 $\pm$ 0.8 nM and 13.0 $\pm$ 0.5 nM at elevated temperature (25 °C, Table 1). Similarly, the dissociation of eIF4G/IRE and eIF4G(eIF4E/IRE complex in the presence of iron was lower at lower temperature (Table 1). The binding data shows that iron enhanced the binding of eIF4G and eIF4G(eIF4E with IRE at all temperatures (Table 1).

To examine the thermodynamic parameters for the binding of ferritin mRNA to eIF4G and eIF4G$\cdot$eIF4E complex with and without addition of iron, temperature dependence equilibrium dissociation constant values were used. The equilibrium data was analyzed by the van’t Hoff equation. Fig. 9 shows the lnK${}_{\mathrm{\text{eq}}}$ vs T${}^{-1}$ plots for the binding of ferritin IRE with eIF4G and eIF4G(eIF4E in the absence and presence of iron. Thermodynamic parameters enthalpy and entropy change calculated using linear fitting of the plot. Data in Table 2 showing that thermodynamic parameters, $\mathrm{\Delta }$H, $\mathrm{\Delta }$S, and $\mathrm{\Delta }$G changes significantly for ferritin IRE RNA/eIFs complexes with the addition of iron. van’t Hoff equation yielded the $\mathrm{\Delta }$H values for the association of eIF4G and eIF4G(eIF4E with IRE-RNA were –42.6 $\pm$ 3.3 kJ. mol${}^{-1}$ and –54.6 $\pm$ 4.3 J. mol${}^{-1}$K${}^{-1}$, respectively, whereas association is entropy opposed for both these complexes. Addition of iron to eIF4G(IRE-RNA significantly changes the $\mathrm{\Delta }$H and $\mathrm{\Delta }$S to –38.1 $\pm$ 3.4 kJ. mol${}^{-1}$ and 34.5 $\pm$ 2.5 kJ. mol${}^{-1}$, whereas for eIF4G$\cdot$eIF4E IRE RNA complex to –45.1 $\pm$ 2.5 kJ. mol${}^{-1}$ and 17.2 $\pm$ 0.7 kJ. mol${}^{-1}$, respectively. The binding free energy value was reported at 25 °C. Interestingly, $\mathrm{\Delta }$G value for the association of ferritin IRE with eIF4G or eIF4G$\cdot$eIF4E complex increased significantly with addition of iron. We observed that the binding free energy and enthalpy changes are predominantly negative sign, favoring association of protein with mRNA. The sign and magnitude of thermodynamic parameters are primarily contributed for the involvement of binding forces between protein and RNA [37]. –$\mathrm{\Delta }$G values suggested that the complex formation at four different temperatures was feasible and the binding reaction was spontaneous. The negative $\mathrm{\Delta }$H value characterized the binding reaction as exothermic. Large negative $\mathrm{\Delta }$H value for the complex formation suggested the involvement of hydrogen bonds [37, 38]. On the other hands a positive $\mathrm{\Delta }$S value was obtained for the eIF4G/IRE and eIF4G(eIF4E/IRE in the presence of iron indicated involvement of hydrophobic interactions. Therefore, our data concluded that the main acting forces are hydrogen bonds as well as hydrophobic interactions in eIF4G/IRE-RNA and eIF4G(eIF4E/IRE-RNA complex formation. Binding free energy, $\mathrm{\Delta }$G, for the eIF4G/IRE-RNA and eIF4G(eIF4E/IRE-RNA with addition of iron change to –48.4 $\pm$ 4.2 kJ. mol${}^{-1}$ and –50.2 $\pm$ 2.7 kJ. mol${}^{-1}$, respectively, which corresponds to the change in the $\mathrm{\Delta }$G of binding by about 8.5 and 9.2, typical value for two additional noncovalent hydrogen bonds [39]. Enhancement in $\mathrm{\Delta }$G of binding for eIF4G(IRE RNA and eIF4G(eIF4E-IRE RNA in the presence of iron, suggest that Fe${}^{2+}$ promotes structural alterations in eIF4G/IRE and eIF4G(eIF4E/IRE-RNA complexes, allowing stable complex formation with translation initiation factors, subsequently upregulating protein synthesis.

Fig. 9.

Thermodynamics of ferritin IRE RNA interaction with the initiation factors described by the van’t Hoff plot for eIF4G (—$\mathrm{\circ }$—), eIF4G-Fe${}^{\mathrm{2}\mathrm{+}}$ (—$\mathrm{\bullet }$—), eIF4G$\mathrm{\cdot }$eIF4E (—$\mathrm{△}$—), and eIF4G$\mathrm{\cdot }$eIF4E-Fe${}^{\mathrm{2}\mathrm{+}}$ (—$\mathrm{▲}$—), respectively.