Cellular iron level regulates the expression of iron encoded
proteins by iron responsive elements (IREs) mRNA at
5-untranslated region (UTR). IREs are present in either the 3-UTR or 5-UTR.
Based on the IREs location at untranslated region of target mRNA, iron regulatory
proteins (IRPs) act as a post-transcriptional modulator of mRNA encoding protein
[1, 2]. Ferritin protein synthesis requires the interaction of stem-loops
structures of IREs-mRNA in the 5-UTR with IRP1. 30-nts stem-loop structure of
IREs is highly conserved. The stem is stabilized by upper helix base pairing and
a pseudo-tri loop with sequence CAGUGX (C denoted A, C or U), lower helix
cytosine bulge and an asymmetric loop [1, 3]. Iron can have opposite metabolic
effect; acts as either translational enhancer or repressor based on the location
of IRE either 3- or 5-UTR . Studies reported that removal of the IREs
stem-loop structure leads to the IRP independent translation . Disturbance in
cellular iron concentration can destabilizes IRE-IRP interaction which leads to
either enhanced translation or inhibit translation of iron regulatory, iron
transport, and iron storage proteins . Therefore, the elevated iron levels in
brain cells destabilize the IRE-dependent signaling pathway which directly or
indirectly contributes to the Alzheimer’s amyloid precursor protein aggregation
and neuronal loss in Alzheimer’s diseases [6, 7]. Iron balance is critical at
cellular level as well as systemic level and mis-regulation of iron can lead to
oxidative damage and cell death including Alzheimer’s disease, Parkinson’s
disease, Friedreich’s ataxia, multiple sclerosis and neuroferritinopathy [8, 9, 10].
It has been reported that high iron in the brain can cause several disorders in
the central nervous system. Elevated level of iron has been observed in the brain
of Alzheimer’s patients [11, 12]. This high iron accumulation in the amyloids of
Alzheimer’s patients leads to disrupt the complex formation between iron
responsive elements and iron regulatory proteins, which leads to the alterations
in the storage and transport of iron [5, 13].
Translation initiation in eukaryotes requires the concerted effort of several
eIFs. Among the protein complexes required is the cap binding complex, eIF4F.
Initiation include series of events that initiate with the interaction of 5-cap
moiety of mRNA with eIF4F [14, 15]. Eukaryotic translation initiation factor eIF4F
is comprised of subunits eIF4E, eIF4G, and eIF4A. Interaction of RNA with eIF4E
and eIF4G are the key factors for initiation of protein synthesis. The small
subunit eIF4E interacts with 5-mRNA cap structure; eIF4A unwinds secondary
structure of mRNA at 5-UTR to promote the binding of ribosome . The subunit
eIF4G is a large multidomain protein interacts with several other translation
initiation factors on the 5-leader sequences to promote scanning of 40S ribosome
to the correct AUG initiation codon and start mRNA translation [17, 18].
Functional domains of eIF4G contains several binding sites for RNA and other eIFs
[14, 19, 20], which is largely responsible for ribosome attachment, mRNA
circularization and enhancing efficiency of mRNA translation through many protein
and RNA interactions. Previously we have shown that IRE RNA binds to eIF4F and
IRP1 protein [4, 21, 22, 23]. Further iron enhances the interactions between eIF4F
with IRE RNA and simultaneously increases translation via inhibiting IRP/IRE RNA
interactions. To further examine the role of eIF4G in translation, we
investigated the interaction between ferritin IRE RNA and eIF4G and correlate the
binding affinity with translational efficiency. We demonstrated that ferritin IRE
RNA strongly binds to eIF4G but not eIF4E. Moreover, addition of exogenous eIF4G
in depleted wheat germ lysate significantly enhanced ferritin IRE mRNA
translation in vitro.
2. Materials and Methods
30-nucleotide (sequence: 5-GUUCUUGCUUCAACAGUGUUUGAACGGAAC-3) ferritin IRE mRNA
and 5S RNA 30-nt (5-UAGUACUUGGAUGGGAGACCGCCUGGGA AU-3) 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 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 . Capped mRNA was
synthesized by using the Ribo mG 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.
2.2 In Vitro Translation Assay
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 L) was mixed to
mGTP-Sepharose (300 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 . 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 . Briefly, 50 (l reaction mixture included 10
g ferritin IRE-luc mRNA template; 25 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 (50 micro M). When
Fe was used, experiments were anaerobic conditions. Nitrogen-purged
solution of 0.1 M HCl used to dissolve FeSO as described previously .
After addition of the luciferase assay reagent (100 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.
2.3 Fluorescence Measurements
The titration experiments for the ferritin IRE RNA interaction with eIF4G, eIF4E
or eIF4GeIF4E complex were performed by fluorescence intensity
measurements as previously described . Protein fluorescence was excited at
280 nm, and the data was collected at of
340 nm. Titration experiments were performed for 0.1 M eIF4G,
eIF4E, or eIF4GeIF4E 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, 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
(T 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
eIF4GeIF4E with ferritin IRE RNA were determined by measuring the
intrinsic fluorescence intensity of eIFs protein and protein-IRE RNA solution.
Normalized fluorescence (F/F) between eIFs-IRE
complex and individual eIFs intrinsic fluorescence intensity were used to measure
the dissociation constant (K) as described previously [30, 31]. In
control experiments, fluorescence intensity of eIFs (0.1 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 . 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
2.4 Binding Sites Measurements
The number of IRE binding site on the eukaryotic initiation factor eIF4G were
measured by exciting the protein and protein-RNA complex at
280 nm and fluorescence intensity was recorded at emission
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 – 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], where [eIF4G] 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 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).
2.5 Competitive Binding Studies
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 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.
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
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].
2.6 Thermodynamic Parameters from van’t Hoff Analysis
Temperature dependence of dissociation constant were used to construct van’t
Hoff plots according to the equation:
where K is the binding affinity at each temperature (5, 10, 15,
20, and 25 °C). The entropy changes S and enthalpy change
H were calculated using the intercept and slope of lnK versus temperature (T). The change in free energy G
of the binding reaction was calculated at 25 °C by the following
3.1 eIF4G-Dependent Translation of Ferritin IRE mRNA
We have previously  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
. eIF4F-depleted lysate was generated by binding of WG extract to
mGTP-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.
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 was 50 M. Point
represents the average of three experiments, and the error bars are indicated.
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 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 in DWG lysate supplemented with eIF4G restored translation by 90% for
capped/uncapped mRNA by stabilizing eIF/IRE RNA complex  and destabilizing
IRP1/IRE RNA complex to promote translation . 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
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 eIF4GIRE 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.
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
(——) and uncapped (——) ferritin mRNA in depleted
wheat germ extract supplemented with eIF4G; for capped (——) and
uncapped (——) ferritin mRNA translation in depleted wheat germ
extract supplemented with eIF4E.
3.2 Interaction of Ferritin IRE with eIF4G
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) and number of binding site (n) of IRE RNA to eIF4G
was measured by the slope and intercept of a Scatchard plot (Q/IRE]
10 vs Q). The K and n for the interaction of
ferritin IRE to eIF4G were 14.8 10 M and 1.0. These
results suggest that eIF4G contain one binding domain for IRE RNA.
K values of ferritin IRE with eIF4G were calculated by non-linear
least squares method (K = 63.0 nM) and Scatchard plot. Equilibrium
values are reported in K and K, whereas
K = 1/K. The results obtained by two-independent
data analysis methods are in good agreement.
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, = 280 nm,
= 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
eIF4GeIF4E. eIF4G binds the ferritin IRE RNA with the
dissociation constant (K) of 63 4.3 nM (Fig. 6), similar
to the binding of beta-globin mRNA  and several fold higher K
than ferritin IRE RNA/eIF4F binding . 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 . Strong binding of IRE-RNA with eIF4G, yet
without the mG cap, explains the earlier examination that removing
mG-cap from ferritin IRE does not affect the translation compared to the
larger change on removing the IRE stem-loop structure . 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 (eIF4GIRE RNA-Fe,
K = 17.0 nM; eIF4GIRE RNA, K =
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.
Ferritin IRE RNA tightly binds to eIF4G. The normalized
fluorescence values for the binding of eIF4G (——),
eIF4GeIF4E (——), and eIF4E (——) versus
concentration of ferritin IRE RNA. eIF4G (——) did not bind a 5S RNA
30-oligonucleotide stem loop used as a negative control. eIF4G, eIF4E, and
eIF4GeIF4E concentration were 100 nM.
Iron enhances the ferritin IRE binding to eIF4G. Histogram
representation of affinity constant (KM) of
ferritin IRE with eIF4E, eIF4G, and eIF4GeIF4E 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
eIF4GeIF4E 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 . Fluorescence results revealed that
ferritin IRE and eIF4E have different binding site on eIF4G.
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 (——), 50 nM eIF4E (——), and
100 nM eIF4E (——). Parallel lines of Lineweaver-Burk plots indicate
3.3 Temperature Dependence Ferritin IRE/eIF4G Binding
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 eIF4GeIF4E/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
eIF4GeIF4E/IRE complex enhanced with increased temperature.
K values increased from 18.2 1.1 nM to 63 4.3 nM and 9.1 0.3
nM to 46.3 3.6 nM for the binding of eIF4G and eIF4GeIF4E with
IRE-RNA as temperature elevated from 5 °C to 25 °C (Table 1).
Fluorescence result revealed that dissociation of eIF4G/IRE and
eIF4GeIF4E/IRE at 5 °C were higher as compared to 25
°C (Table 1). Addition of iron (50 M Fe) shows that eIF4G and
eIF4GeIF4E binding to ferritin IRE increased by about 4-time.
Dissociation constant for the eIF4G/IRE and eIF4G(eIF4E/IRE with addition of
Fe increased to 17.0 0.8 nM and 13.0 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).
Table 1.Temperature dependence binding affinity of ferritin IRE RNA
interaction with eIF4G and eIF4GeIF4E determined by fluorescence
3.4 Thermodynamics of Ferritin IRE Binding to eIF4G
To examine the thermodynamic parameters for the binding of ferritin mRNA to
eIF4G and eIF4GeIF4E 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 vs T 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, H,
S, and G changes significantly for ferritin IRE RNA/eIFs
complexes with the addition of iron. van’t Hoff equation yielded the H
values for the association of eIF4G and eIF4G(eIF4E with IRE-RNA were –42.6
3.3 kJ. mol and –54.6 4.3 J. molK,
respectively, whereas association is entropy opposed for both these complexes.
Addition of iron to eIF4G(IRE-RNA significantly changes the H and
S to –38.1 3.4 kJ. mol and 34.5 2.5 kJ.
mol, whereas for eIF4GeIF4E IRE RNA complex to –45.1
2.5 kJ. mol and 17.2 0.7 kJ. mol, respectively. The binding
free energy value was reported at 25 °C. Interestingly, G
value for the association of ferritin IRE with eIF4G or eIF4GeIF4E
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 . –G values suggested that the
complex formation at four different temperatures was feasible and the binding
reaction was spontaneous. The negative H value characterized the
binding reaction as exothermic. Large negative H value for the complex
formation suggested the involvement of hydrogen bonds [37, 38]. On the other hands
a positive 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, G, for the eIF4G/IRE-RNA and
eIF4G(eIF4E/IRE-RNA with addition of iron change to –48.4 4.2 kJ.
mol and –50.2 2.7 kJ. mol, respectively, which corresponds
to the change in the G of binding by about 8.5 and 9.2, typical value
for two additional noncovalent hydrogen bonds . Enhancement in G of
binding for eIF4G(IRE RNA and eIF4G(eIF4E-IRE RNA in the presence of iron,
suggest that Fe promotes structural alterations in eIF4G/IRE and
eIF4G(eIF4E/IRE-RNA complexes, allowing stable complex formation with translation
initiation factors, subsequently upregulating protein synthesis.
Thermodynamics of ferritin IRE RNA interaction with the
initiation factors described by the van’t Hoff plot for eIF4G
(——), eIF4G-Fe (——),
eIF4GeIF4E (——), and
eIF4GeIF4E-Fe (——), respectively.
Table 2.Thermodynamic data of ferritin IRE binding to eIF4G and
eIF4GeIF4E determined by fluorescence titrations.
This report for the first time shows the specificity of eIF4G by which ferritin
IRE mRNA interact and stimulates capped/uncapped translation of structured mRNA.
Iron increases the binding affinity of ferritin IRE to eIF4G, whereas decreases
the ferritin IRE binding to IRP [4, 21]. Thus, eIF4G-dependent IRE-mRNA
translation elevates. To be able to respond quickly to any cellular iron changes,
upregulation/repression of mRNA must change rapidly. Previous  report showed
the eIF4F binds to IRE, and this binding performs crucial role for translation
initiation of ferritin mRNA. Recent data reported , eIF4F/IRE binding
correlates with the translational efficiency of ferritin mRNA. Berset et
al.  showed that initiation factor eIF4G have three domains for RNA binding.
If eIF4G domain responsible for RNA binding deleted, it directly affects the
in vitro and in vivo translation efficiency and binding
affinity. Translation of capped/uncapped ferritin mRNA in eIF4F-depleted WG
lysates showed more efficient in the presence of eIF4G compared to eIF4E, with
the larger effect attributable to capped mRNA. Addition of eIF4G significantly
enhances translation of ferritin luciferase reporter mRNA in depleted WG lysate.
We show that ferritin mRNA was unable to bind with eIF4E. Addition of increasing
amount of eIF4E did not influence the translation of ferritin mRNA in
eIF4F-dependent wheat germ lysate. These results reveal that supplementation of
eIF4G in depleted WG lysate restored translation, suggesting the involvement of
eIF4G binding to ferritin IRE mRNA for protein synthesis.
To further understand these interactions, quantitative measurements were made
between ferritin IRE mRNA and two subunits eIF4G and eIF4E. It has been shown
that IRE binds with eukaryotic translation initiation factor eIF4F [4, 31], but
specificity of eIF4G binding and eIF4G-dependent translation efficiency had not
been investigated. Present study provides direct evidence for the interaction of
eIF4G with ferritin IRE RNA. Ferritin IRE RNA strongly binds to eIF4G as compared
to eIF4E. Binding data showed that eIF4G contain single strong binding affinity
for ferritin IRE. Our recent studies  using intrinsic protein fluorescence
had shown that eIF4F bound to ferritin IRE tighter than eIF4G and noncompetitive
binding for ferritin IRE and eIF4E. Binding data suggest that ferritin IRE forms
complex with eIF4G following m7G cap/eIF4E complex formation, and this assembly
with eIF4E might only involve in the initiation of protein synthesis of ferritin
IRE, possibly 43S pre-initiation complex formation. Recently  interactions
between eIF4F, IRE and mG cap have been demonstrated the non-competitive
binding support the notion of IRE and mG cap binding on eIF4G and eIF4E.
This selective binding to eIF4G was found to functionally drive expression of
ferritin mRNA. This effect appears to depend on eIF4G/IRE interactions.
Temperature-dependent binding constant reveals that eIF4G/IRE RNA and
eIF4GeIF4E/IRE complex formation is enthalpy driven and entropy
opposed. The strong interaction between initiation factor 4G and IRE is related
with high H of association (–42.6 3.3 kJ.mol). Presence
of iron significantly changes the H, S, and G
values for the interaction of eIF4G and eIF4GeIF4E with ferritin IRE.
Iron enhances the affinity of eIF4G and eIF4GeIF4E complex for IRE RNA
about 4-fold; the G value increased ~8.5 kJ.mol
and 9.2 kJ.mol for the eIF4G and eIF4GeIF4E/IRE with addition
of iron. This change in G (5–6 kJ.mol) indicates the formation
of additional H-bond, and salt bridge between protein and RNA molecule [39, 41].
These data show that additional H-bonds further stabilized the ferritin IRE
RNA/eIF4G complex formation. It has been reported that alteration of H-bonding
involves overall conformation change of molecule . eIF4G subunit responsible
for the interaction with RNA through H-bonding, salt bridge, and van der Waals
forces [20, 43, 44]. This RNA binding along with ribosomal attachment promotes
efficient translation. eIF4E subunit directly interacts with mRNA cap and
promotes initiation events through conformational change of eIF4E .
Interaction of ferritin IRE with eIF4F induces structural alteration in
eukaryotic initiation factor 4F . Consequently, these structural changes
provide favorable positioning for stable complex formation for an effective
eIF4G-dependent translation. Previously [21, 46] reported that iron enhanced
interaction of ferritin IRE with eIF4F, while increase dissociation of IRP from
IRP/IRE-RNA complex, and subsequent enhance translation by forming more stable
platform for further assembly of the initiation factors. Iron increases eIF4G/IRE
complex binding affinity and facilitates assembly of other initiation factor
binding for efficient mRNA translation. Taken together, these data provide
insights into how eIF4G interacts with ferritin IRE and facilitates
eIF4G-dependent translation. There are still much to be learned about the role of
other initiation factors in iron translation regulation.
Ferritin IREs stem-loop structure specifically
interacts with eIF4G subunit of eIF4F. eIF4G binding to the IRE RNA is enhanced
by iron. Both equilibrium and thermodynamic of eIF4G binding to IRE-mRNA are
controlled by iron and promote eIF4G-dependent protein synthesis. Thus, when free
cellular iron levels enhance, rate of ferritin protein synthesis enhances more
than the housekeeping protein, aconitase, because when iron levels lower, a
larger fraction of ferritin IRE-mRNA molecules were inactivated by IRP interact
than aconitase IRE molecules. Cellular iron ion destabilizes mRNA/IRP complexes
competes with the stabilization conferred by hydrogen bonds between IRE RNA and
eIF4G. Iron enhances the binding affinity of eIF/IRE complex and facilitates
translational efficiency by forming a more stable platform for further assembly
point of other initiation factor, demonstrating biological importance of this
binding during cellular environment changes.
IRE, iron responsive element; IRP, iron regulatory protein; WG, wheat germ; DWG,
depleted wheat germ; eIF, eukaryotic initiation factor; UTR, untranslated region.
MAK conceived and designed the experiments; performed the experiments; analyzed
the data; contributed reagents and materials; wrote the paper.
Ethics Approval and Consent to Participate
I thank Research, Innovation and Graduate Council, and College of Science,
Alfaisal University for providing all necessary facilities.
This study was supported by grant from the Alfaisal University Research support,
Riyadh, Saudi Arabia (IRG20413 to MAK).
Conflict of Interest
The author declares no conflict of interest.