The domains of yeast eIF4G, eIF4E and the cap fine-tune eIF4A activities through an intricate network of stimulatory and inhibitory effects

Abstract

Translation initiation in eukaryotes starts with the recognition of the mRNA 5′-cap by eIF4F, a hetero-trimeric complex of eIF4E, the cap-binding protein, eIF4A, a DEAD-box helicase, and eIF4G, a scaffold protein. eIF4G comprises eIF4E- and eIF4A-binding domains (4E-BD, 4A-BD) and three RNA-binding regions (RNA1–RNA3), and interacts with eIF4A, eIF4E, and with the mRNA. Within the eIF4F complex, the helicase activity of eIF4A is increased. We showed previously that RNA3 of eIF4G is important for the stimulation of the eIF4A conformational cycle and its ATPase and helicase activities. Here, we dissect the interplay between the eIF4G domains and the role of the eIF4E/cap interaction in eIF4A activation. We show that RNA2 leads to an increase in the fraction of eIF4A in the closed state, an increased RNA affinity, and faster RNA unwinding. This stimulatory effect is partially reduced when the 4E-BD is present. eIF4E binding to the 4E-BD then further inhibits the helicase activity and closing of eIF4A, but does not affect the RNA-stimulated ATPase activity of eIF4A. The 5′-cap renders the functional interaction of mRNA with eIF4A less efficient. Overall, the activity of eIF4A at the 5′-cap is thus fine-tuned by a delicately balanced network of stimulatory and inhibitory interactions.

INTRODUCTION

The initiation of translation on eukaryotic mRNAs depends on a number of translation initiation factors (eIFs). These factors cooperate in recognizing the 5′-end of the mRNA by binding to its 5′-cap, in recruitment of the 40S ribosomal subunit to the 5′-end, and in scanning of the pre-initiation complex (PIC) along the 5′-untranslated region (5′-UTR) to the AUG start codon. When the start codon is reached, the 60S ribosomal subunit is recruited, and protein synthesis commences (reviewed in (1–4)).

The initial recognition of the mRNA 5′-end involves the translation factor eIF4F, a hetero-trimeric complex formed by the assembly of eIF4A, eIF4E, and eIF4G (5,6). eIF4E is the cap-binding protein that specifically binds to the 5′-cap structure (7,8). eIF4E also interacts with the scaffold protein eIF4G (5,9,10). eIF4G in turn binds to eIF4A (5,11), a DEAD-box helicase that has been implicated in the unwinding of double-stranded regions in the 5′-UTR that might otherwise interfere with PIC recruitment and scanning (12). eIF4A is a much more efficient helicase when part of the eIF4F complex (13–15). eIF4B, and in mammals eIF4H, are RNA binding proteins that further stimulate eIF4A activities in translation initiation (15–22).

The cellular levels of translation initiation factors are regulated according to the physiological conditions (23,24); in humans, their dysregulation is linked to cancer (25). The concentrations of individual translation initiation factors in yeast have been determined under standard growth conditions (26). These studies revealed that eIF4E and eIF4A are present in excess over eIF4G (2 μM), making eIF4G the limiting factor for eIF4F formation. The affinities of eIF4G for eIF4A and eIF4E are in the low micromolar and nanomolar range, respectively (27–29). Thus, under cellular conditions, eIF4G is present almost exclusively as part of the eIF4F complex. The eIF4A/4G complex, although not significantly populated in equilibrium, might be populated transiently as an intermediate during eIF4F assembly (29). Notably, most of eIF4A and eIF4E is left without binding partners, available for other tasks outside eIF4F and independent of the 5′-cap. While eIF4E also participates in mRNA export from the nucleus (30), the cellular function of free eIF4A is less clear (31).

The basic interaction principles within the eIF4F complex can be gleaned from crystal structures and NMR analyses of yeast and human eIF4E in the apo form and in complex with the 5′-cap, and structures of Saccharomyces cerevisiae and human eIF4E/4G and eIF4A/4G complexes (9–11,32–36). The interaction between eIF4E and eIF4G is largely conserved between yeast and humans, despite the substantially different domain organizations of yeast and human eIF4G. The eIF4G/4E complex is formed through the interaction of eIF4E with the eIF4E-binding domain (4E-BD) of eIF4G (10,37,38) (Figure 1A). In humans, a non-canonical loop of eIF4G contacts the lateral side of eIF4E; this loop is absent in yeast eIF4G (10). The interaction of eIF4E with the 4E-BD of 4G leads to an increase in the affinity of eIF4E for the 5′-cap (27,39,40). In yeast, this increase in affinity has been ascribed to an allosteric effect of 4E-BD binding to the surface distal from the cap-binding site of eIF4E (9). For human eIF4E, the affinity is increased through binding of multiple RNA-binding domains of eIF4G to mRNA regions outside the cap, which anchors eIF4E on the mRNA (41). Notably, the interaction of yeast eIF4E with the 5′-cap varies tremendously from mRNA to mRNA, with association rate constants covering more than an order of magnitude (42). The velocity of association is anti-correlated with the propensity of the mRNA to form secondary structures (42).

Figure 1.

Translation initiation factors and RNA substrates used. (A) eIF4A, eIF4B, eIF4E and eIF4G constructs used in this work. eIF4G-ERA: deletion variant of eIF4G comprising amino acids 512–952; eIF4G-RA: deletion variant of eIF4G comprising amino acids 492–952; eIF4G-A: deletion variant of eIF4G comprising amino acids 572–952 (previously termed eIF4G-MC). eIF4G-R*A comprises the same domains as eIF4G-RA, but arginines 493, 497, 504, 509, 514, 519, 523, 528, 529, 532, 537 and 538 in RNA2 have been exchanged to alanines (48). Note that all eIF4G variants also contain the third, C-terminal RNA-binding region (RNA3) not reflected in their names. 4A-BD: eIF4A-binding domain; 4E-BD: eIF4E-binding domain; bio: biotinylation sequence; His₆: hexa-histidine tag; NTD, CTD: N-, C-terminal domain; PABP: poly-A-binding protein binding domain; r1-7: 7-repeats domain; RBD: RNA-binding domain; RecA_N, RecA_C: N- and C-terminal RecA domain; RNA2: second RNA-binding region of eIF4G; RNA3: third RNA-binding region of eIF4G; RRM: RNA recognition motif. The stars denote the cysteines C186 and C370 for donor/acceptor labeling of eIF4A. (B) RNAs used in this work. The blue sphere indicates the fluorescein attached to the 5′-end of the 10mer for fluorescence detection. The yellow sphere represents the 5′-cap. cap-50mer: capped 50mer.

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The interaction of eIF4A with eIF4G is mediated by the eIF4A-binding domain (4A-BD) of 4G (Figure 1A), both in yeast and humans (43,44). Human eIF4G contains a second eIF4A binding site (43,45–47). Both sites are crucial for translation initiation (45), but only the site conserved between humans and yeast is involved in the stimulation of the eIF4A ATPase activity (46). Through this site, eIF4G binds to both RecA-like domains of eIF4A and pre-aligns them in a half-open state (11,28,32,35), poised for formation of the closed state that causes duplex destabilization and RNA unwinding (11,28). Both human and yeast eIF4G also contain several RNA-binding regions that mediate interactions with the mRNA (14,48). Yeast eIF4G contains three regions involved in RNA binding, termed RNA1 to RNA3 (Figure 1A). The most N-terminal region, RNA1, has been implicated in the assembly of the eIF4F/mRNA complex, whereas RNA2, located between the 4E-BD and the 4A-BD, and RNA3, located in the C-terminal region of eIF4G, are thought to play a role in downstream processes such as PIC attachment and scanning (49). Deletion of RNA1 leads to a moderate, approx. two-fold decrease in the rate constant for RNA unwinding by eIF4F, whereas deletion of RNA2 or RNA3 reduces the rate constant for unwinding by 10–15-fold, and abrogates the preference of eIF4F for RNA substrates with a 5′-single-stranded tail (14).

The DEAD-box helicase eIF4A and eIF4E do not interact directly, but affect one another through their association with eIF4G and the mRNA: Yeast eIF4A accelerates the association of eIF4E with the cap (42). This effect correlates with the propensity of the mRNA for structure formation, and could be an indirect effect caused by the helicase function of eIF4A. In turn, human eIF4E stimulates RNA unwinding by eIF4A within the eIF4F complex (13).

Despite a large body of biochemical data (e.g. (13–18,21,28,44,50–57)), the regulatory interplay between the different factors and their constituent domains in cap-dependent and -independent interactions with mRNA is not very well understood. We have previously shown that the RNA3 domain of eIF4G contributes to the stimulation of the ATPase activity of eIF4A by increasing k_cat and decreasing K_1/2 for RNA, and to the promotion of eIF4A closing by eIF4G (18). Here, we analyze the interplay between RNA2 and the 4E- and 4A-BDs of eIF4G in eIF4A activation, and dissect the role of eIF4E and the 5′-cap for eIF4A activities. We show that the presence of RNA2 is correlated with an increase in the fraction of eIF4A in the closed state, an increased RNA affinity, and faster RNA unwinding. This stimulatory effect is partially reduced when the 4E-BD is present. eIF4E and the 5′-cap show independent effects on eIF4A: eIF4E binding to eIF4A/4G is associated with a reduced unwinding activity and a smaller fraction of eIF4A in the closed state, but does not alter the eIF4A ATPase activity and its functional interaction with RNA. The 5′-cap has little effect on RNA unwinding or ATP hydrolysis by eIF4A, but leads to an increase in the K_1/2 value for RNA, indicating that the functional interaction of capped RNA with eIF4A is less efficient. Overall, the activity of eIF4A at the 5′-cap is fine-tuned by a delicately balanced network of stimulatory and inhibitory effects.

MATERIALS AND METHODS

Protein production and purification

Yeast eIF4A, N-terminally biotinylated eIF4A_Q186C/G370C (bio-eIF4A), eIF4E, and the eIF4G variants eIF4G-A, -RA, -R*A and -ERA (Figure 1A) were produced recombinantly in Escherichia coli. eIF4G-A (amino acids 572–952) comprises the middle and C-terminal domains of eIF4G, i.e. the eIF4A binding domain (4A-BD) and RNA3. It was previously termed eIF4G-MC (16–18,51), but now renamed to eIF4G-A to indicate the presence of the 4A-BD. eIF4G-RA and -R*A (amino acids 492–952) both comprise RNA2, the 4A-BD and RNA3; in eIF4G-R*A, twelve arginines within RNA2 are replaced by alanines (in positions 493, 497, 504, 509, 514, 519, 523, 528, 529, 532, 537 and 538), which abrogates RNA binding (48). eIF4G-ERA (amino acids 380–952) comprises the eIF4E-binding domain (4E-BD), the second of three RNA-binding domains (RNA2), the 4A-BD, and RNA3. eIF4A, eIF4A-bio, eIF4B and eIF4G-A were purified as described (17,18). eIF4G-ERA, -RA, and -R*A were overproduced in E. coli Rosetta (DE3) in 2YT medium at 37°C. Gene expression was induced with 0.1 mM isopropyl-β-d-thiogalactoside (IPTG) at an optical density at 600 nm (OD₆₀₀) of 0.6, and cell growth was continued overnight at 20°C. eIF4E was overproduced in E. coli BL21 (DE3) in LB medium. Gene expression was induced with 0.1 mM IPTG at OD₆₀₀ = 0.6 and continued for 4 h at 37°C. Cells overproducing eIF4E, eIF4G-ERA, -RA, or R*A were harvested by centrifugation and resuspended in 50 mM Tris/HCl pH 7.5, 500 mM NaCl, 20 mM imidazole and 2 mM β-mercaptoethanol (β-ME) and disrupted in a microfluidizer. The supernatant was applied to a Ni²⁺-NTA sepharose column (GE Healthcare, 10 ml), and eluted with the same buffer supplemented with 200 mM imidazole. The His₆-tag (eIF4E, eIF4G-RA, -R*A) or His₆-SUMO-tag (eIF4G-ERA) was cleaved with thrombin (eIF4E) or TEV protease (eIF4G-ERA, eIF4G-RA, eIF4G-R*A) during overnight dialysis against 50 mM Tris/HCl pH 7.5, 500 mM NaCl and 2 mM β-ME at 4°C. The tags and uncleaved fusion protein were removed in a second purification step on Ni²⁺-NTA sepharose. The flowthrough was concentrated to < 5 mL and further purified on an S75 (eIF4E) or S200 (eIF4G-ERA, -RA, -R*A) size-exclusion column (120 ml, GE Healthcare) equilibrated with 50 mM Tris/HCl pH 7.5, 500 mM NaCl and 2 mM β-ME. eIF4G-ERA was further purified on a MonoQ ion-exchange column (1 ml, GE Healthcare) equilibrated in 50 mM Tris/HCl pH 7.5, 100 mM NaCl and 2 mM β-ME. After a washing step with 5 % buffer containing 50 mM Tris/HCl pH 7.5, 1 M NaCl, and 2 mM β-ME, 4G-ERA was eluted in a linear gradient from 5 to 20% of the high-salt buffer in 25 ml. Protein purity was >95% according to Coomassie-stained SDS-PAGE.

The plasmid for overproduction of the capping enzyme was provided by Remco Sprangers (University of Regensburg, Germany). The capping enzyme was produced and purified as described (58). The plasmid for overproduction of T7 RNA polymerase Y639F was provided by Roland K. Hartmann (University of Marburg, Germany). T7 RNA polymerase was produced and purified as described (59).

Pull-down assays

Interactions of eIF4G variants with eIF4A and eIF4E were tested in pull-down experiments using His₆-tagged eIF4E. To this end, 0.5 μM His₆-eIF4E were incubated with 2 μM eIF4A and 0.2 μM eIF4G in 40 mM Tris/HCl pH 7.5, 300 mM NaCl, 10 mM MgCl₂, 20 mM glycerol and 0.2 mM Tween20 on ice for 1 h. Complexes formed were isolated by incubation with 20 μl Ni²⁺-NTA resin per 100 μl at 25°C for 1 h; the sample was constantly mixed during the incubation. The resin was precipitated by centrifugation (800 g, 30 s). As a control for unbound protein, 15 μl of the supernatant were removed. The resin was washed three times with 500 μl of 50 mM Tris/HCl pH 7.5, 500 mM NaCl, 20 mM imidazole, and 2 mM β-ME. Complexes were eluted with the same buffer supplemented with 200 mM imidazole, followed by centrifugation (800 g, 30 s). 15 μl of the supernatant were analysed by SDS-PAGE on 15 % acrylamide gels.

DNA and RNA oligonucleotides

50mer RNA (5′-GGG GAG AAA AAC AAA ACA AAA CAA AAC AAA ACU AGC ACC GUA AAG CAC GC; the sequence to which the 10mer to generate the double-stranded substrate binds is underlined) for unwinding assays was purchased HPLC-purified from Dharmacon, the complementary 10mer (5′-GCU UUA CGG U) and 10mer labeled with fluorescein at the 5′-end (5′-FAM-GCU UUA CGG U) were purchased HPLC-purified from Sigma. For capping purposes, ATPase activity assays, and single-molecule FRET experiments, the 50mer was produced by in vitro-transcription from a linear DNA template (5′-CAG AGA TGC ATA ATACGA CTC ACT ATAG GGG GAG AAA AAC AAA ACA AAA CAA AAC AAA ACT AGC ACC GTA AAG CAC GC; the T7 RNA polymerase binding site is underlined, the transcription start is highlighted in bold). The two DNA strands used to generate the DNA template in vitro transcription were purchased from Sigma and annealed by heating an equimolar mixture to 95°C for 1 min and slow cooling to room temperature.

In vitro transcription and capping of RNA

50 ng/μl DNA template were incubated with 0.01 volumes of T7 RNA polymerase and 0.12 U inorganic pyrophosphatase (ThermoFisher) in 40 mM HEPES/KOH pH 7.5, 2 mM dithiothreitol (DTT), 5 mM spermidine, 5% (v/v) dimethyl sulfoxide, 40 mM MgCl₂ and 4 mM NTPs (each) for 2 h at 37°C in 50 μl aliquots. After further addition of 1/75 volume of T7 RNA polymerase, the reaction was continued for 2 h at 37°C. Reactions were stopped with 45 mM EDTA. The RNA was precipitated by adding 1/10 volume of 3 M sodium acetate and 2.5 volumes of 100% ethanol at −20°C, and pelleted by centrifugation (21 000 g, 30 min, 4°C). The pellet was washed twice with 70% ethanol and dried. The RNA was dissolved in ddH₂O, and purified by electrophoresis on an 8 M urea polyacrylamide gel, and extracted from the gel by crush elution and precipitation.

For subsequent capping, 1 nmol of RNA was incubated in ddH₂O at 65°C for 10 min and stored at 4°C for 5 min. The capping reaction was carried out with 0.5 mM GTP, 0.1 mM S-adenosyl-methionine, and 1/10 volume of capping enzyme in 50 mM Tris/HCl pH 8.0, 5 mM KCl, 1 mM MgCl₂ and 1 mM DTT for 1 h at 37°C, followed by RNA precipitation.

RNA binding

Binding of eIF4G-A, -RA and R*A to the 50/10mer RNA was followed in fluorescence anisotropy titrations of the 50/10mer (formed by annealing the 50mer with the 5′-FAM-labeled 10mer) with the respective eIF4G variant in 30 mM HEPES/KOH, pH 7.5, 100 mM KOAc, 3 mM Mg(OAc)₂, and 2 mM DTT at 25°C in a Jobin-Yvon Fluoromax-4 spectrometer equipped with polarizers. Fluorescence was excited at 490 nm (3 nm bandwidth) and parallel and perpendicular components of the emission at 520 nm (6 nm bandwidth) were measured for 3 min to calculate the fluorescence anisotropy.

RNA unwinding

50 μM 50mer RNA or 30 μM capped 50mer RNA and 25 μM or 15 μM of 10mer-fluorescein RNA were annealed in 25 mM HEPES/KOH pH 7.5 by incubating for 2 min at 96°C, slow cooling down to room temperature and incubating on ice for 15 min. 500 nM of this double-stranded RNA were incubated with 5 μM of 10mer (as a trap) and 5 μM eIF4A in the presence and absence of 5 μM eIF4G, 5 μM eIF4E, 5 μM eIF4B and 3 mM ATP. Reactions were performed in 50 mM Tris/HCl pH 7.5, 80 mM KCl, 2.5 mM MgCl₂, 1 mM DTT and 1% (v/v) glycerol at 25°C. The reaction was stopped at different time points with 1% SDS, 50 mM DTT, 5% (v/v) glycerol, 0.05 mg/ml proteinase K. Substrate and product were separated by native gel electrophoresis on a 15% acrylamide gel in TBE. Band intensities were quantified by densitometry, and the fraction unwound at each time point was determined from the ratio of substrate and product. Data were described by single-exponential functions to determine the rate constant k_unwind.

Steady-state ATPase activity

The ATPase activity of eIF4A was determined in a coupled enzymatic assay which couples ATP hydrolysis to NADH oxidation (28,60). Concentrations were 1 μM eIF4A, 2 μM eIF4E, 2 μM eIF4G, 2 μM eIF4B, 2 mM ATP, 0.4 mM NADH, 1 mM phosphoenolpyruvate, 23 μg/ml pyruvate kinase, 13 μg/ml lactate dehydrogenase, and 0 to 200 mM (bases of) RNA. Reactions were performed in 50 mM Tris/HCl pH 7.5, 80 mM KCl, 2.5 mM MgCl₂, 1 mM DTT and 1% (v/v) glycerol at 30°C in a transparent PS F-boden 384 well plate (Greiner bio-one) in a TECAN M1000 pro microplate reader. Reaction velocities were determined from the slopes of the absorbance curves. The dependence of the reaction velocities on the RNA concentration was described with the Michaelis-Menten equation to determine the k_cat for ATP hydrolysis and K_1/2 values for RNA.

Fluorescence labeling and single-molecule FRET experiments

Bio-eIF4A carrying cysteines in RecA_N (Q186C) and RecA_C (G370C) was labeled with a 2-fold molar excess of Alexa555-maleimide (A555, donor) and a 5-fold molar excess of Alexa647-maleimide (A647, acceptor). The labeling efficiency was determined from the absorption at 280, 554 and 647 nm.

For single-molecule FRET experiments, 5× activity buffer (250 mM Tris/HCl pH 7.5, 400 mM KCl, 12.5 mM MgCl₂, 5 mM DTT and 5% (v/v) glycerol) was treated with active charcoal overnight and filtered through a 0.4 μM membrane. All experiments were performed in activity buffer.

Experiments were performed with 150 pM labeled bio-eIF4A (donor concentration) in a BSA-saturated microwell slide (A10657-01, Hamamatsu) on a Picoquant Microtime 200 confocal microscope in the presence and absence of 8 μM eIF4G-A, 8–10 μM eIF4G-RA, 8 μM eIF4G-R*A, or 6 μM eIF4G-ERA, 6 μM eIF4E, 10 μM 50mer RNA (molar concentration) or 1 mM poly-U-RNA (concentration in bases), and 3 mM ATP at 25°C. Fluorescence intensities were corrected for donor crosstalk in the acceptor channel, acceptor crosstalk in the donor channel, different quantum yields and detection efficiencies of donor and acceptor fluorescence and direct excitation of the acceptor as previously described (61).

RESULTS

We previously showed that RNA3 of yeast eIF4G contributes to the stimulation of the conformational cycle of eIF4A and its ATPase and helicase activities (17,18). Here, we used a set of eIF4G deletion variants (Figure 1A) to further investigate the role of eIF4G domains in eIF4A activation: eIF4G-ERA comprises the 4E-BD, two of the three RNA binding domains (RNA2 and RNA3) and the eIF4A binding domain (4A-BD), and interacts with eIF4E and eIF4A (Supplementary Figure S1A,B). A shorter variant, eIF4G-RA, comprises RNA2, the 4A-BD and RNA3, but lacks the 4E-BD. This variant interacts with eIF4A, but not with eIF4E (Supplementary Figure S1B,C). Finally, eIF4G-A comprises the 4A-BD and RNA3, but lacks the 4E-BD and RNA2. This variant was termed eIF4G-MC in previous studies (16–18,51). As eIF4G-RA, eIF4G-A interacts with eIF4A, but not with eIF4E (Supplementary Figure S1A).

RNA2 plays a role in promoting eIF4A closing, its interaction with RNA, and RNA unwinding

To delineate the effect of RNA2 on RNA unwinding, we compared RNA unwinding activities of eIF4A in the presence of eIF4G-RA and 4G-A (Figure 2A, B), using a 50/10mer substrate employed previously to characterize eIF4A activities (16,17,51,62). This RNA substrate contains an unstructured 5′-single-stranded tail that mimics an unstructured 5′-UTR (Figure 1B). Reactions in the absence of ATP were performed as a negative control (Supplementary Figure S2). The low intrinsic helicase activity of eIF4A, with a rate constant of unwinding of k_unwind = 0.15 (± 0.03) × 10⁻³ s⁻¹, is stimulated both by eIF4G-A and eIF4G-RA (Figure 2A, B, Table 1). Consistent with previous observations (11,17,18), eIF4G-A binding increases the rate constant of unwinding by eIF4A about 4-fold, to k_unwind = 0.6 (± 0.1) × 10⁻³ s⁻¹. In the presence of eIF4G-RA, unwinding is stimulated by 13-fold, with k_unwind = 1.9 (± 0.2) × 10⁻³ s⁻¹ (Figure 2A, B, Table 1, Supplementary Figure S3A). These values correspond to a 3-fold stimulation of unwinding by RNA2. It is important to note that the stimulation factors contain contributions from stimulatory effects caused by the eIF4G variant present and by eIF4B. If eIF4B and 4G act cooperatively in stimulating RNA unwinding by eIF4A, with different levels of cooperativity for eIF4G-A and RA, these values constitute upper limits. The unwinding amplitudes are similar (∼70%) with both eIF4G variants (Figure 2B, Supplementary Figure S3B).

$Effect of RNA2 on RNA unwinding, ATP hydrolysis and eIF4A conformation. (A) Representative unwinding reactions of 0.5 μM 50/10mer by eIF4A (5 μM) in the presence of 5 μM eIF4B and 5 μM eIF4G-A, eIF4G-RA or 4G-ERA in 50 mM Tris/HCl pH 7.5, 80 mM KCl, 2.5 mM MgCl2, 1 mM DTT and 1% (v/v) glycerol. ss: single-stranded 10mer RNA, ds: double-stranded 50/10mer RNA. On the right-hand side, a schematic of the unwinding assay is shown; the blue sphere indicates the fluorescein attached to the 5′-end of the 10mer for fluorescence detection. Unwinding experiments were performed at least twice and gave similar results (see Supplementary Figure S3C). (B) Fraction of RNA unwound as a function of time, determined from densitometric quantification of single- and double-stranded RNA, for eIF4A (gray), eIF4A and eIF4G-A (blue), eIF4A and eIF4G-RA (green) or eIF4A and eIF4G-ERA (red). Error bars reflect the SEM from duplicate experiments. Rate constants for unwinding and reaction amplitudes are summarized in Table 1. See Supplementary Figure S2 for controls in the absence of ATP. (C) ATPase activity of 1 μM eIF4A in the presence of 50mer RNA (concentrations as indicated), 2 μM eIF4B and 2 μM eIF4G-ERA (red), eIF4G-RA (green) or eIF4G-A (blue) in 50 mM Tris/HCl pH 7.5, 80 mM KCl, 2.5 mM MgCl2, 1 mM DTT, and 1% (v/v) glycerol. Error bars reflect the SEM from duplicate experiments. (D) Single-molecule FRET histograms of eIF4A in the presence of 50mer RNA and eIF4G-ERA, eIF4G-RA or eIF4G-A. In the presence of eIF4G-A, 25 ± 4% of eIF4A molecules are in the closed conformation, 76 ± 8% in the presence of eIF4G-RA and 61 ± 8% in the presence of eIF4G-ERA. Errors are the SEM from duplicate experiments. See Table 3 for a summary of the fraction closed. Green: half-open conformation of eIF4A with a FRET efficiency of 0.25, blue: closed conformation with a FRET efficiency of 0.70. FRET efficiencies below zero (red, marked by gray rectangles) result from donor-only labeled species. The cyan curve represents a fit of the sum of three Gaussian distributions to the data. Histograms show representative data; experiments were performed at least twice and gave similar results (see Supplementary Figure S6).$

Figure 2.

Effect of RNA2 on RNA unwinding, ATP hydrolysis and eIF4A conformation. (A) Representative unwinding reactions of 0.5 μM 50/10mer by eIF4A (5 μM) in the presence of 5 μM eIF4B and 5 μM eIF4G-A, eIF4G-RA or 4G-ERA in 50 mM Tris/HCl pH 7.5, 80 mM KCl, 2.5 mM MgCl₂, 1 mM DTT and 1% (v/v) glycerol. ss: single-stranded 10mer RNA, ds: double-stranded 50/10mer RNA. On the right-hand side, a schematic of the unwinding assay is shown; the blue sphere indicates the fluorescein attached to the 5′-end of the 10mer for fluorescence detection. Unwinding experiments were performed at least twice and gave similar results (see Supplementary Figure S3C). (B) Fraction of RNA unwound as a function of time, determined from densitometric quantification of single- and double-stranded RNA, for eIF4A (gray), eIF4A and eIF4G-A (blue), eIF4A and eIF4G-RA (green) or eIF4A and eIF4G-ERA (red). Error bars reflect the SEM from duplicate experiments. Rate constants for unwinding and reaction amplitudes are summarized in Table 1. See Supplementary Figure S2 for controls in the absence of ATP. (C) ATPase activity of 1 μM eIF4A in the presence of 50mer RNA (concentrations as indicated), 2 μM eIF4B and 2 μM eIF4G-ERA (red), eIF4G-RA (green) or eIF4G-A (blue) in 50 mM Tris/HCl pH 7.5, 80 mM KCl, 2.5 mM MgCl₂, 1 mM DTT, and 1% (v/v) glycerol. Error bars reflect the SEM from duplicate experiments. (D) Single-molecule FRET histograms of eIF4A in the presence of 50mer RNA and eIF4G-ERA, eIF4G-RA or eIF4G-A. In the presence of eIF4G-A, 25 ± 4% of eIF4A molecules are in the closed conformation, 76 ± 8% in the presence of eIF4G-RA and 61 ± 8% in the presence of eIF4G-ERA. Errors are the SEM from duplicate experiments. See Table 3 for a summary of the fraction closed. Green: half-open conformation of eIF4A with a FRET efficiency of 0.25, blue: closed conformation with a FRET efficiency of 0.70. FRET efficiencies below zero (red, marked by gray rectangles) result from donor-only labeled species. The cyan curve represents a fit of the sum of three Gaussian distributions to the data. Histograms show representative data; experiments were performed at least twice and gave similar results (see Supplementary Figure S6).

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Table 1.

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Rate constants and amplitudes of unwinding reactions

		+ eIF4G-ERA		+ eIF4G-RA		+ eIF4G-A		− eIF4G
		k_unwind	ampl.	k_unwind	ampl.	k_unwind	ampl.	k_unwind	ampl.
		(10⁻³ s⁻¹)	(%)	(10⁻³ s⁻¹)	(%)	(10⁻³ s⁻¹)	(%)	(10⁻³ s⁻¹)	(%)
uncapped	- 4E	1.3 ± 0.2	72 ± 10	1.9 ± 0.2	67 ± 4	0.6 ± 0.1	66 ± 6	0.15 ± 0.03	n.d.
	+ 4E	0.53 ± 0.06	74 ± 1	1.4 ± 0.4	78 ± 6	0.5 ± 0.2	69 ± 6
capped	- 4E	1.3 ± 0.1	79 ± 15	1.7 ± 0.2	93 ± 2	0.58 ± 0.07	61 ± 13	0.11 ± 0.0004	n.d.
	+ 4E	0.9 ± 0.3	84 ± 11	1.31 ± 0.07	90 ± 3	0.7 ± 0.1	75 ± 8

		+ eIF4G-ERA		+ eIF4G-RA		+ eIF4G-A		− eIF4G
		k_unwind	ampl.	k_unwind	ampl.	k_unwind	ampl.	k_unwind	ampl.
		(10⁻³ s⁻¹)	(%)	(10⁻³ s⁻¹)	(%)	(10⁻³ s⁻¹)	(%)	(10⁻³ s⁻¹)	(%)
uncapped	- 4E	1.3 ± 0.2	72 ± 10	1.9 ± 0.2	67 ± 4	0.6 ± 0.1	66 ± 6	0.15 ± 0.03	n.d.
	+ 4E	0.53 ± 0.06	74 ± 1	1.4 ± 0.4	78 ± 6	0.5 ± 0.2	69 ± 6
capped	- 4E	1.3 ± 0.1	79 ± 15	1.7 ± 0.2	93 ± 2	0.58 ± 0.07	61 ± 13	0.11 ± 0.0004	n.d.
	+ 4E	0.9 ± 0.3	84 ± 11	1.31 ± 0.07	90 ± 3	0.7 ± 0.1	75 ± 8

Errors denote SD from triplicate experiments for 4A/4G-ERA/4E + capped RNA and for 4A/4G-A/4E + capped RNA. In all other cases, SEM from duplicate experiments are given; n.d.: not determined.

Table 1.

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Rate constants and amplitudes of unwinding reactions

		+ eIF4G-ERA		+ eIF4G-RA		+ eIF4G-A		− eIF4G
		k_unwind	ampl.	k_unwind	ampl.	k_unwind	ampl.	k_unwind	ampl.
		(10⁻³ s⁻¹)	(%)	(10⁻³ s⁻¹)	(%)	(10⁻³ s⁻¹)	(%)	(10⁻³ s⁻¹)	(%)
uncapped	- 4E	1.3 ± 0.2	72 ± 10	1.9 ± 0.2	67 ± 4	0.6 ± 0.1	66 ± 6	0.15 ± 0.03	n.d.
	+ 4E	0.53 ± 0.06	74 ± 1	1.4 ± 0.4	78 ± 6	0.5 ± 0.2	69 ± 6
capped	- 4E	1.3 ± 0.1	79 ± 15	1.7 ± 0.2	93 ± 2	0.58 ± 0.07	61 ± 13	0.11 ± 0.0004	n.d.
	+ 4E	0.9 ± 0.3	84 ± 11	1.31 ± 0.07	90 ± 3	0.7 ± 0.1	75 ± 8

		+ eIF4G-ERA		+ eIF4G-RA		+ eIF4G-A		− eIF4G
		k_unwind	ampl.	k_unwind	ampl.	k_unwind	ampl.	k_unwind	ampl.
		(10⁻³ s⁻¹)	(%)	(10⁻³ s⁻¹)	(%)	(10⁻³ s⁻¹)	(%)	(10⁻³ s⁻¹)	(%)
uncapped	- 4E	1.3 ± 0.2	72 ± 10	1.9 ± 0.2	67 ± 4	0.6 ± 0.1	66 ± 6	0.15 ± 0.03	n.d.
	+ 4E	0.53 ± 0.06	74 ± 1	1.4 ± 0.4	78 ± 6	0.5 ± 0.2	69 ± 6
capped	- 4E	1.3 ± 0.1	79 ± 15	1.7 ± 0.2	93 ± 2	0.58 ± 0.07	61 ± 13	0.11 ± 0.0004	n.d.
	+ 4E	0.9 ± 0.3	84 ± 11	1.31 ± 0.07	90 ± 3	0.7 ± 0.1	75 ± 8

Errors denote SD from triplicate experiments for 4A/4G-ERA/4E + capped RNA and for 4A/4G-A/4E + capped RNA. In all other cases, SEM from duplicate experiments are given; n.d.: not determined.

The presence of RNA2 also had an effect on the ATPase activity of eIF4A (Figure 2C): In the presence of eIF4G-A, eIF4A hydrolyzed ATP with a turnover number of k_cat = 83 (± 1) × 10⁻³ s⁻¹. The stimulation of the ATPase activity by RNA is governed by a K_1/2 = 270 ± 15 nM. Both values are in agreement with previous reports (17,18). In the presence of eIF4G-RA containing RNA2, the turnover number was increased slightly, to k_cat = 97 (± 3) × 10⁻³ s⁻¹, at the same time K_1/2 for RNA was slightly decreased to K_1/2 =212 ± 20 nM (Table 2A). Thus, RNA2 enhances the functional interaction of eIF4A with RNA.

We have shown previously that eIF4B also promotes functional RNA interactions with eIF4A, manifested as an approx. three-fold reduction on the K_1/2 value of eIF4A for RNA in the presence of eIF4B (16,18). We therefore tested whether the stimulatory effect of RNA2 is also present in the absence of eIF4B (Supplementary Figure S4A, B, Table 2B). Notably, the moderate effect of RNA2 observed in the presence of eIF4B becomes more pronounced when eIF4B is absent, with a three-fold decrease in K_1/2 (Table 2B).

eIF4A switches between different conformational states during ATP hydrolysis and RNA unwinding, which can be distinguished by their different FRET efficiencies in single-molecule FRET experiments (18,28). To test whether RNA2 has an effect on eIF4A conformation, we performed single-molecule FRET experiments with donor-acceptor-labeled eIF4A in the presence of eIF4G-A and eIF4G-RA (Figure 2D). In the presence of 50mer and ATP, we detected two different FRET states of eIF4A bound to eIF4G-A or eIF4G-RA: one with a FRET efficiency E_FRET of approx. 0.25, corresponding to the half-open state observed previously (28), and a second with E_FRET of approx. 0.70, corresponding to the closed state (18) (Figure 2D). These observations demonstrate that eIF4G-RA also stabilizes eIF4A in a half-open state, similar to eIF4G-A, and promotes eIF4A closing in the presence of ATP and RNA. The relative populations of the two states of eIF4A in the presence of eIF4G-A and eIF4G-RA differ, though: With eIF4G-A, 25 ± 4% of the eIF4A molecules are in the closed state. With eIF4G-RA this fraction is increased approx. three-fold, to 76 ± 8% (Figure 2D, Table 3), showing that the presence of RNA2 is associated with the promotion of the conformational change of eIF4A to the closed state. To test whether the increased capacity of eIF4G-RA to stimulate closing of eIF4A is caused by the interaction of RNA2 with RNA, we purified eIF4G-R*A, a construct comprising RNA2, the 4A-BD, and RNA3, in which twelve arginines within RNA2 are exchanged to alanines. These mutations had been reported to abrogate RNA binding to RNA2 (48). Anisotropy titrations demonstrate that mutation of these arginines has a similar deleterious effect on RNA binding as deletion of RNA2 (Supplementary Figure S5A). Single-molecule experiments with donor/acceptor-labeled eIF4A in the presence of eIF4G-R*A, poly-U-RNA, and ATP showed a significantly reduced fraction of eIF4A in the closed state compared to experiments in the presence of eIF4G-RA, similar to the observations with eIF4G-A lacking RNA2 (Supplementary Figure S5B). Altogether, the stimulatory effect of RNA2 on closing of eIF4A, its functional interaction with RNA, and its unwinding activity is thus linked to RNA binding to RNA2.

The 4E-BD partially reverts the stimulatory effects of RNA2

We next tested the effect of the 4E-BD of eIF4G on eIF4A activities, first in the absence of eIF4E (Figure 2). In the presence of eIF4G-ERA, eIF4A unwound the 50/10mer with a rate constant of k_unwind = 1.3 (± 0.2) × 10⁻³ s⁻¹ (Figure 2A, Table 1). This value corresponds to a stimulation of its intrinsic unwinding activity by 9-fold, which is less than the stimulation achieved with eIF4G-RA (13-fold). Similarly, the fraction of eIF4A in the closed conformation was increased by eIF4G-ERA (fraction closed = 61 ± 8%; Figure 2D, Table 3), but less than with eIF4G-RA (76 ± 8%). Thus, the 4E-BD reduces the favorable effect of RNA2 on unwinding and closing of eIF4A. In contrast, the K_1/2 value of eIF4A for RNA, K_1/2 =173 ± 30 nM, was slightly lower in the presence of eIF4G-ERA compared to eIF4G-RA (K_1/2=212 ± 20 nM; Figure 2C, Table 2A), in agreement with an additional, positive effect of the 4E-BD on the interaction of eIF4A with RNA. This effect was also observed in the absence of eIF4B (Table 2B). The k_cat values were similar in the absence and presence of the 4E-BD (Table 2A, 2B).

Table 2A.

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K_1/2 and k_cat values for RNA-dependent ATP hydrolysis by eIF4A in the presence of eIF4B and different eIF4G variants, and effects of eIF4E and the 5′-cap

		+ eIF4G-ERA		+ eIF4G-RA		+ eIF4G-A
		K_1/2	k_cat	K_1/2	k_cat	K_1/2	k_cat
		(nM)	(10⁻³ s⁻¹)	(nM)	(10⁻³ s⁻¹)	(nM)	(10⁻³ s⁻¹)
uncapped	− 4E	173 ± 30	99 ± 1	212 ± 20	97 ± 3	270 ± 15	83 ± 1
	+ 4E	199 ± 28	100 ± 3	193 ± 39	92 ± 14	457 ± 38	82 ± 1
capped	− 4E	304 ± 6	81 ± 1	342 ± 31	92 ± 4	553 ± 193	89 ± 9
	+ 4E	294 ± 41	74 ± 4	374 ± 56	88 ± 6	975 ± 27	89 ± 3

		+ eIF4G-ERA		+ eIF4G-RA		+ eIF4G-A
		K_1/2	k_cat	K_1/2	k_cat	K_1/2	k_cat
		(nM)	(10⁻³ s⁻¹)	(nM)	(10⁻³ s⁻¹)	(nM)	(10⁻³ s⁻¹)
uncapped	− 4E	173 ± 30	99 ± 1	212 ± 20	97 ± 3	270 ± 15	83 ± 1
	+ 4E	199 ± 28	100 ± 3	193 ± 39	92 ± 14	457 ± 38	82 ± 1
capped	− 4E	304 ± 6	81 ± 1	342 ± 31	92 ± 4	553 ± 193	89 ± 9
	+ 4E	294 ± 41	74 ± 4	374 ± 56	88 ± 6	975 ± 27	89 ± 3