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 (K = 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 K
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 H =
–42.6 3.3 kJ. mole, S = –11.5 0.4 J.
moleK, and G = –39.2 2.7 kJ. mole,
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.