eIF2A, an initiator tRNA carrier refractory to eIF2α kinases, functions synergistically with eIF5B
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The initiator tRNA (Met-tRNA i Met ) at the P site of the small ribosomal subunit plays an important role in the recognition of an mRNA start codon. In bacteria, the initiator tRNA carrier, IF2, facilitates the positioning of Met-tRNA i Met on the small ribosomal subunit. Eukarya contain the Met-tRNA i Met carrier, eIF2 (unrelated to IF2), whose carrier activity is inhibited under stress conditions by the phosphorylation of its α-subunit by stress-activated eIF2α kinases. The stress-resistant initiator tRNA carrier, eIF2A, was recently uncovered and shown to load Met-tRNA i Met on the 40S ribosomal subunit associated with a stress-resistant mRNA under stress conditions. Here, we report that eIF2A interacts and functionally cooperates with eIF5B (a homolog of IF2), and we describe the functional domains of eIF2A that are required for its binding of Met-tRNA i Met , eIF5B, and a stress-resistant mRNA. The results indicate that the eukaryotic eIF5B–eIF2A complex functionally mimics the bacterial IF2 containing ribosome-, GTP-, and initiator tRNA-binding domains in a single polypeptide.
KeywordsTranslation initiation eIF2A eIF5B Evolution of initiator tRNA carriers
Prokaryotic initiation factor 2
Eukaryotic initiation factors
Eukaryotic initiation factor 2A
Eukaryotic initiation factor 5B
- Initiator tRNA
Met-tRNA i Met
Protein kinase double-stranded RNA-dependent
PKR-like ER kinase
General control non-derepressible-2
Internal ribosome entry site
Hepatitis C virus
The translation of an mRNA begins when a small ribosomal subunit finds the initiation codon of an mRNA. The presence of an initiator tRNA (Met-tRNA i Met ) at the P site of the small ribosomal subunit (the 30S and 40S ribosomes of prokaryotes and eukaryotes, respectively) critically enables the recognition of the initiation codon . Translation factors called initiator tRNA carriers facilitate the placement of Met-tRNA i Met onto the P site of small ribosomal subunits. The bacterial translation factor, IF2, is a typical initiator tRNA carrier that facilitates the allocation of a formylmethionine-charged initiator tRNA (fMet-tRNA i fMet ) onto the P site of the 30S ribosomal subunit. To ensure correct placement, IF2: (1) associates with fMet-tRNA i fMet via an interaction between the C-terminal region of IF2 and the formyl group of fMet-tRNA i fMet ; (2) interacts with the 30S ribosome through the N-terminal region of IF2 ; (3) facilitates the joining of the large ribosomal subunit (the 50S ribosome) with the 30S ribosome ; and concomitantly (4) hydrolyzes GTP and dissociates from the 30S ribosome [5, 6].
Eukaryotic IF2 (eIF2) is the major initiator tRNA carrier responsible for loading eukaryotic Met-tRNA i Met onto the 40S ribosomal subunit. eIF2 is composed of three polypeptides, the α, β, and γ subunits. Of them, the γ subunit has GTPase and Met-tRNA i Met -binding activities ; the β subunit facilitates the association of eIF2 and the 40S ribosome through an interaction with eIF3 ; and the α subunit regulates the activity of eIF2 . More specifically, the phosphorylation of a regulatory serine (serine 51) of the eIF2α subunit (in yeast) inhibits the Met-tRNA i Met carrier activity of eIF2 via the sequestration of eIF2B, which is the guanosine nucleotide exchange factor responsible for the GDP–GTP exchange activity of eIF2 [10, 11]. A number of eIF2α kinases are activated by various stress signals, including oxidative stress [heme-regulated inhibitor (HRI) or EIF2AK1], viral infection [protein kinase double-stranded RNA-dependent (PKR) or EIF2AK2], ER overload [PKR-like ER kinase (PERK) or EIF2AK3], and ROS accumulation or amino acid starvation [general control non-derepressible-2 (GCN2) or EIF2AK4]. Under the relevant stress conditions, these kinases phosphorylate the regulatory serine of eIF2α, thereby repressing the translation of most eukaryotic mRNAs . After eIF2 delivers Met-tRNA i Met , the carrier is released from the 40S ribosome through a conformational change induced by GTP hydrolysis . eIF5B, a homolog of IF2, stabilizes the association of initiator tRNA with the 40S ribosome and helps to ensure the correct positioning of Met-tRNA i Met on the P site of the 40S ribosome when the start codon of an mRNA pairs with the anticodon of Met-tRNA i Met [14, 15].
The translations of several mRNAs are known to be refractory to the translational inhibition mediated by the phosphorylation of eIF2α . Numerous studies have sought to understand the molecular basis of the persistent translation of specific mRNAs under stress conditions. Many such studies have focused on identifying the carrier(s) for Met-tRNA i Met in the translation of the hepatitis C viral (HCV) mRNA, which is efficiently translated under stress conditions when the eIF2α subunit is heavily phosphorylated . Several cellular proteins have been suggested to participate in recruiting Met-tRNA i Met to a 40S ribosome associated with an HCV mRNA containing an internal ribosome entry site (IRES) [18, 19, 20, 21]. In addition, eIF2A, which stimulates the GTP-independent binding of Met-tRNA i Met to a 40S ribosome programmed with AUG [22, 23], was reported to be responsible for the persistent translation of HCV mRNA under stress conditions . The authors showed that siRNA-mediated knockdown of eIF2A severely repressed HCV mRNA translation under stress conditions. Moreover, the addition of recombinant eIF2A proteins to an eIF2A-depleted in vitro translation system restored HCV mRNA translation in the presence of phosphorylated eIF2α. eIF2A strongly interacts with Met-tRNA i Met and with stem-loop IIId of the HCV IRES, which was shown to be essential for HCV mRNA translation under stress conditions . The data thus indicate that eIF2A is responsible for supplying Met-tRNA i Met to a 40S ribosome associated with an HCV mRNA, and that the direct interaction of eIF2A with the HCV mRNA is required for its selective translation under stress conditions. Several papers, which suggest that eIF2A functions in translation initiation, have been published after the report. For instance, eIF2A was shown to mediate the stress-resistant translation of c-Src mRNA, which is essential for cell proliferation under stress conditions . The authors demonstrated that eIF2A facilitates tRNA i Met loading onto the 40S ribosome in a c-Src mRNA-dependent manner. Similarly, to the direct interaction of eIF2A with the HCV mRNA, eIF2A directly interacts with c-Src mRNA . The results indicate that the direct interaction of eIF2A with a specific mRNA plays an important role in determining the mRNA specificity of Met-tRNA i Met loading to a 40S ribosome. Interestingly, recent reports suggest that eIF2A participates in translation of mRNAs utilizing CUG codon as a translation start codon [26, 27, 28, 29]. The reports suggested that eIF2A participates in antigen presentation by MHC class 1 molecule, stress responses, and tumor initiation. However, the molecular basis of the activities of eIF2A in translation of these mRNAs has been poorly understood. Curiously, an eIF2A-null mouse has been generated recently, and the eIF2A-null mouse did not show an apparent abnormality under normal growth conditions . It would be interesting if the eIF2A-null mouse shows an abnormality under stress conditions.
A comparison of eIF2A with IF2 reveals functional similarities and differences. Both proteins directly bind to initiator tRNAs and deliver them to small ribosomal subunits in an AUG-dependent manner , thereby differing from eIF2, which delivers Met-tRNA i Met to the 40S ribosome in an AUG-independent manner . Unlike IF2, however, eIF2A neither directly interacts with the small subunit of ribosome nor has the GTPase activity that is required for IF2 and eIF2 to dissociate from the ribosome after they deliver Met-tRNA i Met . The mechanisms how eIF2A deploys Met-tRNA i Met in the P site of the 40S ribosomal subunit and how eIF2A dissociates from the 40S ribosome after delivering Met-tRNA i Met remains to be elucidated. We thus hypothesized that it may work cooperatively with another, GTPase activity-bearing protein, to function as a Met-tRNA i Met carrier.
Here, we set out to identify a protein that functions cooperatively with eIF2A as a Met-tRNA i Met carrier. We focused on a translation initiation factor eIF5B that was shown to be genetically related to yeast eIF2A. A previous study showed that knockout of both eIF2A and eIF5B yields a synthetically sick phenotype in the yeast Saccharomyces cerevisiae, suggesting that these proteins function in the same pathway . As eIF5B has ribosome-binding and GTPase activities that could potentially complement the lack of such activities in eIF2A, we herein investigated the potential cooperative function of these proteins to act as a Met-tRNA i Met carrier.
We first confirmed that eIF2A and eIF5B show a genetic interaction in animals, using Caenorhabditis elegans (C. elegans) as a multicellular model organism. In the eIF2A-null mutant, eIF5B knockdown triggered a severe delay in development, suggesting that eIF5B and eIF2A function in the same pathway of C. elegans (Fig. S1). To further examine the molecular basis for this genetic effect, we investigated the potential physical interaction of these proteins, and identified an interaction between purified eIF2A and eIF5B proteins. We determined the domains in eIF2A required for its interactions with eIF5B, Met-tRNA i Met , and mRNA. Furthermore, we performed experiments, showing that eIF5B augments the activity of eIF2A in loading Met-tRNA i Met onto a 40S ribosome associated with an HCV mRNA. Finally, we analyzed the functional domains of eIF2A associated with eIF5B with respect to those of well-known bacterial fMet-tRNA i fMet carrier IF2 as follows: in bacteria, IF2 itself exhibits initiator tRNA-binding, ribosome-binding, GTPase, and large subunit-joining activities; and in Eukarya, the eIF5B–eIF2A complex possesses the equivalent activities of IF2. In addition, the eIF5B–eIF2A complex has a specific mRNA-binding activity that does not exist in IF2. The results provide insight into the molecular basis of how the eIF5B–eIF2A complex enables the translation of specific mRNAs to occur under stress conditions.
Materials and methods
Cell culture, transfection, and immunoprecipitation
293FT cells were cultivated in Dulbecco’s modified Eagle’s medium (Gibco BRL) supplemented with 10% fetal bovine serum (Clontech). Plasmids were transfected to cells using Lipofectamine (Invitrogen). The transfected cells were cultivated on plates, washed with cold PBS (pH 7.4), and then lysed in 500 μl of ice-cold buffer A [0.1% NP-40, 40 mM HEPES–KOH (pH 7.5), 100 mM KCl, 1 mM EDTA, 10 mM β-glycerophosphate, 10 mM NaF, 2 mM Na3VO4, and 1 mM PMSF]. The solution was sonicated on ice and centrifuged to yield whole-cell extracts (WCEs). For immunoprecipitation, WCEs were incubated with 10 μl of anti-Flag M2 affinity gel (Flag resin; Sigma) at 4 °C for 2 h with constant rotation. The beads were collected and washed four times with the same buffer, and the bead–bound proteins were resolved by SDS-PAGE and analyzed by western blotting.
Purification of recombinant proteins and GST pull-down assay
His-tagged eIF2A was expressed in E. coli strain M15 using plasmid pQE31/His–eIF2A, and the expressed protein was purified as previously described . GST-fused eIF5B and GST-fused eIF2A variants were expressed in E. coli strain Bl21. The GST-fused proteins were purified and GST pull-down assays were performed, both as previously described . The purified proteins were visualized by coomassie blue staining (Fig. S2).
In vitro transcription and pull down with biotinylated RNAs
For the production of biotinylated tRNAs and HCV IRESs, plasmids pRL-CMV/Met-tRNA i Met and pRL-CMV/tRNALeu, which were previously reported , were treated by BstN1, whereas pRL-CMV/HCV IRES was treated by AccI . The linearized DNAs were used as templates for in vitro transcription reactions with T7 RNA polymerase in the presence of 1 mM biotinylated UTP. RNA pull-down experiments were performed using purified GST–eIF2A (1 μg) or eIF2A-overexpressing 293FT cell lysates (3 mg) and biotinylated RNAs (3 μg), as previously described .
40S ribosomes were prepared from HeLa cells as described previously . The amounts of components are as follows: 2 pmol of [32P]tRNA i Met (10,000 c.p.m./pmol), 2.5 pmol of 40S ribosomal subunit, 2 pmol of HCV IRES, and 3 pmol each of eIF2A and eIF5B. The procedures were performed as described previously .
Sucrose density gradient analysis
Polysome profile analyses were performed with the cell extracts of mock- or tunicamycin (2 μg/ml)-treated 293FT cells. Briefly, the cells were treated with cycloheximide (100 μg/ml) for 30 min on ice, and lysed with 1 ml of lysis buffer [300 mM KOAc, 10 mM MgCl2, 50 mM HEPES–KOH (pH 7.5), 100 mM NH4Cl, 5 mM DTT, 0.1 mM EDTA, 0.5% NP-40, and 100 μg/ml cycloheximide]. The sucrose gradients were prepared with polysome buffer [300 mM KOAc, 10 mM MgCl2, 50 mM HEPES–KOH (pH 7.5), 100 mM NH4Cl, 2 mM DTT, and 0.1 mM EDTA]. The cell lysate (500 μg) was resolved on 5–45% sucrose gradients through ultracentrifugation for 3 h at 38,000 rpm in an SW41Ti rotor (Beckman). We collected 0.25 ml fractions via a gradient density fractionator (Brandel) using upward displacement with 60% (w/v) sucrose at a flow rate of 0.25 ml/min. Continuous monitoring was performed at an absorbance of 254 nm using an Econo UV monitor (Bio-Rad). Proteins in every second fraction were methanol-precipitated and analyzed by western blotting. rRNAs in the fractions were analyzed by agarose gel electrophoresis after phenol/chloroform extraction followed by ethanol precipitation, and rRNAs were visualized by ethidium bromide staining.
Genetic interaction between eIF2A and eIF5B
The presence of a genetic interaction between eIF2A and eIF5B was previously uncovered in a yeast knockout study , where the single knockouts of eIF2A and eIF5B yielded mild and moderate slow-growth phenotypes, respectively, whereas double knockout of eIF2A and eIF5B yielded a very severe slow-growth phenotype. The “synthetically sick” phenotype of the double mutant was taken as suggesting that these proteins function in the same biological pathway. In the present study, we first set out to confirm the relationship of eIF2A and eIF5B in animals, using knockdown and knockout experiments in the model organism, C. elegans. Briefly, we characterized a null mutant worm lacking eIF2A (E04D5.1) and examined the effect of eIF5B/iffb-1 knockdown (Fig. S1). More specifically, we monitored the effect of eIF5B knockdown on wild-type (panels 1 and 2 in Fig. S1a) and eIF2A-null worms (panels 3 and 4 in Fig. S1a) at 52 h after hatching. Knockout of eIF2A yielded a very mild slow-development phenotype, as shown in Fig. S1b (compare columns 1 and 3). Knockdown of eIF5B in the wild-type background yielded a slow-development phenotype (compare panel 1 with 2 in Fig. S1a; compare column 1 with 2 in Fig. S1b). Importantly, knockdown of eIF5B in the eIF2A mutant background yielded a very severe slow-development phenotype (Fig. S1a and S1b), suggesting that eIF5B and eIF2A work together in the same pathway.
Physical interaction between eIF2A and eIF5B
The middle domain of eIF2A (residues 462–501) is necessary and sufficient for the interaction with eIF5B
To determine the eIF2A-binding domain in eIF5B, GST pull-down experiments were performed with purified recombinant proteins (Fig. S4c). eIF5B is composed of four functional domains (domains G to IV) . The domains G and II are responsible for binding to the 40S ribosomal subunit, and the domain IV is connected with domain III via a long α-helix (H12 of 40 Å). The pull-down experiments indicated that the domains G + II + III + IV of eIF5B strongly interacts with eIF2A, and the domain IV and domains III + IV weakly bind to eIF2A (lanes 9, 11, and 12 in Fig. S4c). However, either domain III or domains II + III did not bind to eIF2A (lanes 10 and 13 in Fig. S4c). The results indicate that the domain IV of eIF5B is the minimal domain for the interaction with eIF2A and that the domains G to IV are needed for the full capacity of interaction with eIF2A.
The WD domain of eIF2A interacts with initiator tRNA
eIF5B synergistically augments the ability of eIF2A to load Met-tRNA i Met onto the 40S ribosomal subunit
Both eIF2A and eIF5B are associated with the 40S ribosomal subunit under stress conditions
Comparison of the structural and functional domains of the bacterial fMet-tRNA i fMet carrier, IF2 (Fig. 6b), with those of eIF2A and eIF5B allowed us to predict the relative positions of eIF2A, eIF5B, and Met-tRNA i Met during their interaction (Fig. 6c). In addition, we predicted the eIF2A structure complexed with the 40S ribosomal subunit associated with eIF5B, tRNA i Met , and HCV IRES based on our results (Fig. S4b). The N-terminal part of eIF2A, which is composed of a WD domain associated with Met-tRNA i Met , may localize over the P site of 40S ribosome to load Met-tRNA i Met on the P site. The structure of M domain of eIF2A could not be predicted by an available structure prediction program, but we could speculate the position of M domain through the interactions between eIF2A and eIF5B and between eIF2A and HCV IRES. The domain IV of eIF5B is likely to stretch out over the P site of 40S ribosomal subunit and interact with the M domain of eIF2A (depicted as a dotted line in Fig. S4b). The M domain of eIF2A is likely to position over the P and E sites of 40S ribosomal subunit and is connected to the C domain that is predicted to be composed of two consecutive alpha helices that binds to the IIId domain of HCV IRES (Fig. S4b). According to the cryo-EM structure of HCV IRES associated with the 40S ribosomal subunit, the IIId domain of HCV IRES sticks out over the platform of 40S ribosome . It is plausible that the C-terminal end of C domain of eIF2A touches the extended part of IIId domain where the 40S ribosomal subunit is not associated. With these configurations of 40S ribosome, eIF5B, eIF2A, Met-tRNA i Met , and HCV IRES, all of the components may form a complex through RNA–protein–protein–RNA interactions (Fig. S4b). The interaction between eIF2A and a specific mRNA seems to play a key role in selecting mRNAs refractory to stress-dependent translational repression . Recently, we reported that the stress-resistant translation of c-Src mRNA directed by an IRES element, which is essential for cell proliferation under stress conditions, is also mediated by eIF2A . A direct interaction between eIF2A and c-Src IRES was shown to be essential for eIF2A-mediated translation . Interestingly, both HCV and c-Src IRESs, which are known to use eIF2A as a Met-tRNA i Met carrier, interact with 40S ribosome directly. However, it is not clear whether only the IRES-containing mRNAs utilize eIF2A as a Met-tRNA i Met carrier. Our current investigations on the mRNAs associated with eIF2A under stress conditions with a genome-wide approach revealed that many cellular mRNAs associate with eIF2A under stress conditions (data not shown). The functionalities of the massive interactions between eIF2A and various mRNAs are under investigation.
Our study of the potential functional cooperation between eIF2A and eIF5B revealed that eIF5B synergistically augments the eIF2A-mediated loading of Met-tRNA i Met onto the 40S ribosome (Fig. 4). Interestingly, the start codon is critical for the HCV IRES-dependent loading of Met-tRNA i Met onto the 40S ribosome, which is mediated by eIF2A. A mutant IRES, which contains a substitution mutation of the start codon from AUG to AAA, failed to augment eIF2A-mediated loading of tRNA i Met onto the 40S ribosome, even though the mRNA-independent loading of tRNA i Met remained in the presence of the mutant HCV IRES (Fig. S6). On the contrary, the eIF5B-mediated loading of tRNA i Met onto the 40S ribosome was completely abolished when the mutant HCV IRES was used in the filter-binding assay (Fig. S6b). In addition, the synergistic activation of tRNA i Met loading onto the 40S ribosome by eIF2A and eIF5B (Fig. 4) was abolished when the mutant HCV IRES was used in the filter-binding assay (Fig. S6b). The difference of tRNA i Met -loading capability of these proteins is likely attributed to the binding affinity of these proteins with tRNA i Met . That is, eIF2A but not eIF5B strongly interacts with tRNA i Met . This indicates that the codon–anticodon interaction between the HCV IRES and the Met-tRNA i Met plays an important role in mRNA-dependent loading of Met-tRNA i Met onto the 40S ribosome, which is mediated by either eIF2A or eIF5B. Based on these results and the previous reports, we speculate that eIF5B enables eIF2A to bind ribosomes, while eIF2A enables eIF5B to bind Met-tRNA i Met associated with an mRNA through a codon–anticodon interaction.
Finally, we used sucrose density gradient analyses to examine the dispersion patterns of eIF2A and eIF5B proteins under normal and stress conditions. Under normal conditions, both proteins were found mostly in the top and 40S fractions (fractions 1 and 2 in Fig. 5). The distribution pattern of eIF5B is somewhat surprising, as it has been suggested to promote the correct placement of Met-tRNA i Met , stabilize Met-tRNA i Met at the P site , and facilitate the association of the 60S and 40S ribosomes . Nevertheless, our findings are consistent with the distribution pattern of eIF5B reported previously . It is noteworthy that knockout of eIF5B yields a slow-growth phenotype, suggesting that its activity is not essential for yeast growth under normal conditions . As an alternative explanation for the observed distribution of eIF5B in the sucrose density gradient, we speculate that eIF5B might have been dissociated from the 40S ribosome during the analytic process. This possibility should be re-investigated in the future. Critically, under stress conditions, eIF5B and eIF2A showed much more co-elution with the 40S ribosome (e.g., they were both found in fraction 3 and 4 in tunicamycin-treated cells) (Fig. 5b). Moreover, our results indicate that these proteins are released from the 40S ribosome later than eIF2 under stress conditions (fraction 3 in Fig. 5b). The distribution patterns of eIF2A and eIF5B in the sucrose density gradient analyses were similar under normal and stress conditions. This may indicate that these two proteins are released from the 40S ribosome as a complex, before the joining of the 60S ribosomal subunit, perhaps, via a conformational change of eIF5B induced by its hydrolysis of GTP. However, further investigation is needed to clearly prove that these two proteins associate and dissociate with the 40S ribosome as a complex. Notably, low levels of eIF2A were detected in all tested fractions (Fig. 5b). It is likely to reflect the direct (ribosome- and eIF5B-independent) association of the C domain of eIF2A with mRNAs.
Evolutionary perspective on initiator tRNA carriers (Fig. 6d)
Given what our present results indicate regarding the tight communication between eIF5B and eIF2A, it seems worthwhile to re-examine the general features and relationships among the carriers of Met-tRNA i Met . In the case of bacteria, a single translation factor, IF2, facilitates the loading of fMet-tRNA i fMet on the P site of the small ribosomal subunit. The N-terminal part of IF2 is responsible for interacting with the 30S ribosome and binding GTP, while the C-terminal part, which is connected to the N-terminal domain via a long α-helix, interacts with a formylmethionine-charged initiator tRNA (fMet-tRNA i fMet ) via the formyl group . In addition, IF2 facilitates the joining of the large ribosomal subunit (the 50S ribosome) to the translational pre-initiation complex composed of the 30S ribosome, mRNA, and fMet-tRNA i fMet [43, 44, 45]. The hydrolysis of GTP on IF2, which is activated by the joining of the large ribosomal subunit, occurs concomitantly with the dissociation of IF2 from the 70S ribosomal complex. The hydrolysis of GTP is believed to induce conformational changes in IF2, triggering this dissociation . Archaea express a homolog of IF2 called archaeal IF2 (aIF2, also known as a/eIF5B), which contains GTP- and ribosome-binding domains . Unlike bacterial IF2, however, aIF2 may not interact with Met-tRNA i Met strongly, since the formylation of methionine, which is essential for the interaction with IF2, does not occur in Archaea and Eukarya . Therefore, aIF2 alone may not be able to recruit Met-tRNA i Met to the P site of the 30S ribosome. Instead, Archaea express an alternative Met-tRNA i Met carrier called a/eIF2, the homolog of eukaryotic translation initiation factor eIF2, which recruits Met-tRNA i Met to the small ribosomal subunit . The cooperative action of a/eIF5B and a/eIF2 facilitates the loading of Met-tRNA i Met at the P site of the small ribosomal subunit [39, 46]. The α subunit of a/eIF2 does not undergo kinase-mediated phosphorylation in Archaea; this mode of repression exists only in Eukarya. Indeed, the Eukarya express structural and functional homologs of archaeal a/eIF2 and a/eIF5B, called eIF2 and eIF5B, respectively. eIF5B is a ribosome-dependent GTPase that mediates ribosomal subunit joining . Similar to a/eIF5B, eIF5B contains a G domain and domain II, which confer GTPase and ribosome-binding activities, respectively . In addition, similar to a/eIF5B, eIF5B binds weakly to Met-tRNA i Met , which makes it less likely to function as an initiator tRNA carrier by itself. Instead, the concerted actions of eIF5B and the Met-tRNA i Met carrier, eIF2, facilitate the loading of Met-tRNA i Met to the P site of the 40S ribosome and stabilize the ribosome-tRNA i Met complex. Moreover, eIF5B accelerates the joining of the ribosomal subunits .
eIF2α kinases of various eukaryotic organisms
The authors thank Prof. Sunghoon Kim (Seoul National University, Republic of Korea) for providing us a Gemt-Easy vector containing an initiator tRNA i Met , tRNALeu-coding region.
EK, JHK, and SKJ designed the experiments. EK, JHK, KS, SWA and JK performed the experiments. KYH provided materials. EK, KS, KYH, SL, and SKJ wrote the paper.
This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (No. NRF-2017R1A2B3009902), and by the National Research Foundation of Korea (NRF) Grant funded by the Korean Government (MSIP) (No. NRF-2017R1A5A1015366).
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