Human eukaryotic initiation factor 4E (eIF4E) and the nucleotide-bound state of eIF4A regulate eIF4F binding to RNA

Abstract

During translation initiation, the underlying mechanism by which the eukaryotic initiation factor (eIF) 4E, eIF4A, and eIF4G components of eIF4F coordinate their binding activities to regulate eIF4F binding to mRNA is poorly defined. Here, we used fluorescence anisotropy to generate thermodynamic and kinetic frameworks for the interaction of uncapped RNA with human eIF4F. We demonstrate that eIF4E binding to an autoinhibitory domain in eIF4G generates a high-affinity binding conformation of the eIF4F complex for RNA. In addition, we show that the nucleotide-bound state of the eIF4A component further regulates uncapped RNA binding by eIF4F, with a four-fold decrease in the equilibrium dissociation constant observed in the presence versus the absence of ATP. Monitoring uncapped RNA dissociation in real time reveals that ATP reduces the dissociation rate constant of RNA for eIF4F by ∼4-orders of magnitude. Thus, release of ATP from eIF4A places eIF4F in a dynamic state that has very fast association and dissociation rates from RNA. Monitoring the kinetic framework for eIF4A binding to eIF4G revealed two different rate constants that likely reflect two conformational states of the eIF4F complex. Furthermore, we determined that the eIF4G autoinhibitory domain promotes a more stable, less dynamic, eIF4A-binding state, which is overcome by eIF4E binding. Overall, our data support a model whereby eIF4E binding to eIF4G/4A stabilizes a high-affinity RNA-binding state of eIF4F and enables eIF4A to adopt a more dynamic interaction with eIF4G. This dynamic conformation may contribute to the ability of eIF4F to rapidly bind and release mRNA during scanning.

Keywords: RNA; cooperativity; eIF4A; eIF4E; eIF4F; eIF4G; translation initiation.

Figure 1

Cap-independent role of eIF4E in RNA binding by the cap-binding complex eIF4F.A, cartoon depicting the binding domains in human eIF4G and the truncations used for this work. B, fluorescence polarization assay plots to determine the equilibrium dissociation constant (K_d) of CAA42-FL to eIF4G_682–1599 eIF4A ATP (blue), and eIF4G_557–1599 eIF4A ATP in the absence (red), or the presence of eIF4E (black). The fraction of CAA42-FL bound at different concentrations of proteins is shown with each point representing the mean of three independent experiments. Error bars indicate the standard errors of the mean. eIF, eukaryotic initiation factor; FL, fluorescein labeled.

Figure 2

Effect of eIF4E on affinity and dissociation kinetics of eIF4A for eIF4G.A, fluorescence polarization assay plots used to determine the equilibrium dissociation constant (K_d) of eIF4A-FL for eIF4G_557–1599 in the presence (cyan) or the absence (orange) of saturating eIF4E. The fraction of eIF4A-FL bound at different concentrations of proteins is shown with each point representing the mean of three independent experiments. Error bars indicate the standard errors of the mean. B, fluorescence polarization kinetic plots used to determine the dissociation rates (k₁ and k₂) of eIF4A-FL for eIF4G_557–1599 in the presence (cyan) or the absence (orange) of saturating eIF4E or for eIF4G_682–1599 (gray). Thick lines represent the fit of the data to a double exponential decay model. Data shown are the average of at least three independent experiments. eIF, eukaryotic initiation factor; FL, fluorescein labeled.

Figure 3

Contribution of the nucleotide-bound state of eIF4A on RNA binding by the eIF4F complex.A and B, fluorescence polarization assay plots used to determine the equilibrium dissociation constant (K_d) of CAA42-FL binding to eIF4G_557–1599 eIF4A eIF4E (A) and eIF4G_682–1599 eIF4A (B), in the absence of nucleotide (green), or the presence of saturating amounts of ATP (red) or ADP (blue). The fraction of CAA42-FL bound at different concentrations of proteins is shown with each point representing the mean of three independent experiments. Error bars indicate the standard errors of the mean. We highlight that to provide visual comparisons, the same curves used in Figure 1 to show eIF4G_557–1599 eIF4A eIF4E and eIF4G_682–1599 eIF4A in the presence of ATP (red lines) are reproduced in this figure. eIF, eukaryotic initiation factor; FL, fluorescein labeled.

Figure 4

The nucleotide-bound state of eIF4A controls the dissociation rate constant of RNA binding to eIF4F. Fluorescence polarization kinetic plots used to determine the dissociation rate (k₁) of CAA42-FL for eIF4G_682–1599 eIF4A in the presence (red) or the absence (green) of ATP or for eIF4A in the presence of ATP (gray), measured at 30 °C. Thick lines represent the fit of the data to a single exponential decay model, except for a green line that represents a baseline value. Data shown are the average of at least three independent experiments. eIF, eukaryotic initiation factor; FL, fluorescein labeled.

Figure 5

Proposed model for RNA binding by eIF4F through a cap-independent function of eIF4E and the nucleotide-bound state of eIF4A.A, in the absence of eIF4E, the eIF4E-binding domain stabilizes a conformation of eIF4G that possesses low RNA-binding affinity and low eIF4A helicase activity. The binding of eIF4E stabilizes a conformation of eIF4G that possesses a high RNA-binding affinity and high eIF4A helicase activity. B, the hyperactive truncation of eIF4G_682–1599 without the autoinhibitory eIF4E-binding domain is depicted in the presence of eIF4A and either no nucleotide (left panel) or ATP (right panel). In the absence of nucleotide, RNA binding and release from the eIF4G_682–1599–eIF4A complex are both fast; the dissociation rate of RNA from eIF4G_682–1599–eIF4A is estimated to be greater than ∼100 s⁻¹, with a calculated association rate (∗) of greater than 1 × 10⁸ M⁻¹ s⁻¹ (left panel). In the presence of nucleotide, RNA binding and release from the eIF4G_682–1599–eIF4A complex is slow; the dissociation rate of RNA from eIF4G_682–1599–eIF4A is 0.067 s⁻¹, with a calculated association rate (∗) of 2.1 × 10⁵ M⁻¹ s⁻¹ (right panel). Calculated association rates are determined using equilibrium dissociation constants (Table 1) and observed dissociation rates (Table 2). C, a model to depict possible intermediates during the recruitment of mRNA to the 43S PIC and its subsequent scanning. The eIF4F complex bound to ATP can form a long-lived complex with mRNA. This complex can bind to the 43S PIC to form the 48S PIC. Hydrolysis of ATP and release of phosphate converts the eIF4F complex into a complex that rapidly releases from the mRNA, which can promote the scanning/translocation of the 48S PIC. Binding of a new ATP molecule will convert eIF4F back into a long-lived RNA-binding state, which will arrest scanning of the 48S PIC until the next hydrolysis event. It is unknown whether the hydrolysis of ATP prior to 43S PIC binding enables productive 43S PIC binding (dotted arrow with question mark). eIF, eukaryotic initiation factor; PIC, preinitiation complex.