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Structural insights into eukaryotic ribosomes and the initiation of translation
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Structural insights into eukaryotic ribosomes and the initiation of translation§ Felix Voigts-Hoffmann, Sebastian Klinge and Nenad Ban The initiation of protein biosynthesis entails the ordered assembly of elongation-competent ribosomes, with an initiator tRNA basepaired to an appropriate mRNA start codon. In eukaryotes, this process is more complex than in prokaryotes and involves numerous protein factors that mediate tRNA delivery, mRNA binding, start codon selection and subunit joining. The recent 40S:eIF1, 80S and eIF2:tRNA:GDPNP ternary complex structures provide an initial structural framework toward a molecular understanding of the eukaryotic translation initiation process. Updated homology models of larger initiation complexes provide first insights into the likely arrangements of these higher-order complexes, but also reveal the limits of our current understanding of the eukaryotic translation initiation process. Address Institute of Molecular Biology and Biophysics, ETH Zurich, 8093 Zurich, Switzerland Corresponding author: Ban, Nenad ([email protected])
Current Opinion in Structural Biology 2012, 22:xx–yy This review comes from a themed issue on Proteins Edited by Anders Liljas and Peter Moore
0959-440X/$ – see front matter, # 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.sbi.2012.07.010
Introduction Translation initiation refers to the formation of translation-competent ribosomes with initiator tRNA basepaired to the mRNA start codon at the ribosomal Psite. Bacteria rely on the Shine–Dalgarno sequence of the mRNA to locate the start codon and require just three initiation factors [1]. In contrast, eukaryotic translation initiation relies on a scanning mechanism to locate the start codon and involves 13 core initiation factors, some of which are large, multimeric complexes [2] (Table 1).
§ Funding sources: We acknowledge support by the Swiss National Science Foundation (SNSF), the National Center of Excellence in Research (NCCR) Structural Biology program of the SNSF, and European Research Council grant 250071 under the European Community’s Seventh Framework Programme. S.K. was supported by EMBO and HFSP Fellowships.
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In eukaryotes, initiator tRNA is delivered to the ribosome as a ternary complex (TC) with eIF2 and GTP (Scheme 1 panel 1). Initiation factors eIF1, eIF1A and eIF3 promote the binding of the TC to form a 43S pre-initiation complex (PIC). The 43S PIC binds to the mRNA through its interactions with the mRNA cap-binding complex eIF4 and begins scanning for the start codon (Scheme 1 panel 2). Hydrolysis of eIF2-bound GTP is the key step in translation initiation, which commits the 43S complex to initiation at a particular start codon. It is controlled by eIF5, which stimulates the hydrolysis of eIF2-bound GTP within the initiation complex, and eIF1, which blocks the release of inorganic phosphate (Pi) [3]. (Scheme 1 panel 3) Upon start codon recognition, eIF1 is displaced from the P-site, leading to the release of Pi and dissociation of eIF2, probably in complex with eIF5 [4] (Scheme 1 panel 4). A second GTPase, eIF5B, then mediates subunit joining and displacement of initiation factors. (Scheme 1 panel 5) Hydrolysis of eIF5B-bound GTP is required for dissociation of eIF5B and eIF1A, vacating the A-site for the first elongator tRNA (Scheme 1 panel 6, for details refer to reviews [1,2,5]). The initiation process is regulated primarily at the level of TC formation and mRNA recruitment [1,2,5], and may be modulated through the availability of the large ribosomal subunit [6]. However, the molecular mechanisms of eukaryotic translation initiation remain to be established. The crystal structures of the 40S:eIF1 complex [7] (PDB: 2XZM), the TC [8] (PDB: 3V11) and the 80S ribosome [9] (PDBs: 3U5B–3U5E) provide an opportunity to generate models of initiation intermediates as a starting point for further analysis (Figure 1).
Structures of the eukaryotic ribosome Compared to prokaryotes, the increased complexity of eukaryotic protein synthesis is not only reflected in the initiation process, but also in the architecture of the ribosome itself [1,2,5,7,9,10]. Whereas the core functional regions such as the peptidyl transferase center (PTC) or the decoding site are conserved between domains of life, eukaryotic ribosomes contain many additional rRNA and protein segments, and some regions, such as the beak and the left foot of the small subunit, have been extensively remodeled. Furthermore, eukaryote-specific protein segments engage in extensive protein–protein contacts, while prokaryotic ribosomal Current Opinion in Structural Biology 2012, 22:1–10
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2 Proteins
Table 1 Eukaryotic core initiation factors. Molecular weights were calculated based on the human amino acid sequence Initiation factor
Bacterial homolog
Subunits
eIF1
None
1 (12.7 kDa)
eIF1A
IF1
1 (16.5 kDa)
eIF2
None
eIF2B
None
eIF3
None
a (36.1 kDa) b (38.4 kDa) g (51.1 kDa) a (33.7 kDa) b (39 kDa) g (50.2 kDa) d (57.6 kDa) e (80.4 kDa) a–ma (790 kDa)
eIF4A eIF4B eIF4E eIF4F
None None None None
1 (46 kDa) 1 (69.1 kDa) 1 (25.1 kDa) E, A, G (140.2)
eIF4G
None
1 (175.5 kDa)
eIF4H eIF5 eIF5B
None None IF2
1 (27.4 kDa) 1 (49.2 kDa) 1 (138.8 kDa)
Function Prevents initiation at noncognate codons or in poor context. Functionally related to bacterial IF3 Cooperates with eIF1 in start codon selection, and is involved in the recruitment of eIF5B GTPase that forms a ternary complex (TC) with initiator tRNA and GTP. Plays a key role in start codon selection Nucleotide exchange factor required for re-activation of eIF2
Stimulates binding of ternary complex (TC) and attachment of 43S complexes to mRNA and participates in scanning DEAD-box ATPase and ATP-dependent RNA helicase Enhances activity of eIF4A Binds to the mRNA cap structure Synonym for a complex of eIF4E, eIF4A and eIF4G. Mediates unwinding of mRNA and attachment of 43S complexes Binds to eIF4E, eIF4A, eIF3, PABP, SLIP1 and mRNA and enhances the helicase activity of elF4A Enhances activity of eIF4A, partially homologous to eIF4B GAP and GDI regulating eIF2 activity GTPase that mediates subunit joining
Adapted from [2]. a
eIF3 has 13 subunits (a–m) in mammals, 5 of which (a, b, c, i, g) are universally conserved.
proteins interact mainly with rRNA. This interconnectivity is particularly evident on the solvent-exposed side of the large subunit, where extensions of ribosomal proteins and novel proteins form an intricate network of interactions with the rRNA expansion segments (for a detailed discussion of the eukaryotic ribosome structures, refer to [7,9,10,11]). While some of the eukaryote-specific proteins may function to stabilize the structure of the ribosomal rRNA [10], others may participate directly in the regulation of translation. For instance, small subunit protein rpS6 is phosphorylated downstream of the mammalian target of rapamycin (mTOR) kinase, which integrates stimuli such as growth factor signaling [7]. With the availability of atomic models [7,9,10], the various novel components of the eukaryotic ribosome may now be characterized to determine structural and functional roles (Figure 1).
Binding of eIF1 and eIF1A to the small subunit Together with eIF2, eIF1 and eIF1A are the main determinants of start codon selection. The presence of eIF1 prevents initiation on codons that are noncognate or present in an improper context, such as a short leader sequence. eIF1 also blocks the release of Pi from eIF2– GDP–Pi in the 43S PIC [3] (Scheme 1 panel 2). The eukaryotic ortholog of bacterial IF1, eIF1A, cooperates with eIF1 in start codon selection and is also involved in the recruitment of eIF5B, the GTPase that mediates Current Opinion in Structural Biology 2012, 22:1–10
subunit joining [5]. Furthermore, eIF1 and eIF1A participate in ribosome recycling: Binding of eIF1, eIF1A and eIF3 to recycled 40S subunits leads to the release of deacetylated P-site tRNA and dissociation of mRNA, while the factors remain bound to the 40S, priming it for the next round of initiation [12] (Scheme 1 panel 7). The crystal structure of the eukaryotic 40S subunit in complex with eIF1 (PDB: 2XZM) reveals that eIF1 binds to the interface side of the 40S body, in proximity to the P-site [7] (Figure 1). eIF1 binds to helix 44 (h44) of the small subunit 18S rRNA via a positively charged surface region. Furthermore, several residues of the basic loop (residues 26–30 in Tetrahymena thermophila) are positioned in close proximity to the mRNA channel. In contrast, the region facing toward the P-site tRNA contains acidic and hydrophobic residues. Steric clashes between eIF1 and tRNA in the canonical P-site orientation (PDBs: 2WDL, 2WDK) suggest that the factor may be displaced by initiator tRNA upon formation of cognate start codon interactions. In support of this model, hydroxyl radical probing data indicate that initiator tRNA may be slightly rotated in the 40S:eIF1 complex [13] (Scheme 1 panel 4, Figure 2). Steric hindrance also underlies the anti-association activity of eIF1. Because its binding site overlaps with the position of the highly conserved helix 69 (h69) of the large subunit 26S rRNA in the 80S ribosome, eIF1 will prevent joining of the large subunit to initiation complexes (Figure 1). www.sciencedirect.com
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Structural insights into eukaryotic ribosomes and the initiation of translation Voigts-Hoffmann, Klinge and Ban 3
Figure 1
(a)
central protuberance
(b) rpS19
rpS18 rpS15
RPL5
head
RPL11
rpS25 rpS5 rpS16
beak
RPL36A (L42) RPL13
rpS28
rpS27A (S31)
RPL10
RPLP0
h69 platform
elF1
P-stalk
RPL4
RPL36
rpS14
RPL7A (L8)
shoulder
RPL40
rpS30
rpS3A (S1)
rpS23
RPL9
RPL8 (L2)
RPL23
RPL37A (L43)
rpS24
RPL34
rpS27
rpS6
elF6
rpS13 RPL27 right foot
rpS11
RPL3
RPL30 RPL38
body
rpS8
RPL24 RPL19
RPL22
left foot
(c)
(d) elF2γ
beak
head P-stalk
elF2α
elF2β RACK1
tRNAiMet
40S
60S
EF-Tu body tRNA
Current Opinion in Structural Biology
(a) Crystal structures of the eukaryotic 40S:eIF1 complex (PDB: 2XZM) and (b) the 60S:eIF6 complex (PDBs: 4A17, 4A19) from Tetrahymena thermophila. Ribosomal rRNA is depicted in gray, ribosomal proteins in different colors. Proteins are labeled according to UNIPROT nomenclature, and yeast protein names are indicated in parentheses, where different. (c) Crystal structure of archaeal (Sulfolobussolfataricus) ternary initiation complex aIF2:GDPNP:initiator-tRNA (PDB: 3V11) compared to bacterial (Escherichia coli) elongation factor EF-Tu:GTP:tRNA ternary complex (PDB: 1B23). (d) Crystal structure of the Saccharomyces cerevisiae 80S ribosome (PDBs: 3U5B–3U5E). The 40S subunit in (a) is rotated 1108 clockwise relative to the position in the 80S (d), the 60S subunit in (b) 708 counterclockwise (as seen from the top).
Modeling of eIF1A (PDB: 2OQK) on the 40S based on the bacterial 30S:IF1 complex (PDB: 1HR0) positions it in the A-site, in contact with h44 and h18 of the 18S rRNA and ribosomal proteins rpS23, rpS27A and rpS30 (Figure 2a). Compared to bacterial IF1, eIF1A has an www.sciencedirect.com
additional alpha helix as well as N-terminal tail (NTT) and C-terminal tails (CTT) that inhibit and enhance scanning, respectively [14–16]. In the superposition, the truncated C-terminus of the eIF1A structure is oriented toward the P-site and eIF1, whereas the Current Opinion in Structural Biology 2012, 22:1–10
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4 Proteins
Figure 2
(a)
N
(b)
mRNA C
elF3
elF2α
N
beak
elF1A
elF1 C
elF2β
tRNAiMet
C
elF2α
elF1A
N elF2γ N
C
(c)
elF2β elF2γ
156
elF5 CTD
N 237 elF2β right foot
left foot
elF5 NTD
C elF1
elF2γ Current Opinion in Structural Biology
(a) Homology model of a small subunit pre-initiation complex. The model is based on the crystal structure of the 40S:eIF1 complex (PDB: 2XZM). 40S ribosomal RNA is shown as gray and white spheres, proteins as light blue surfaces. Initiation factors, mRNA and tRNA are shown as cartoons with transparent surface and colored as follows: P-site tRNA (violet), eIF1 (dark blue), eIF1A (yellow), mRNA (red), eIF2b (dark green), eIF2g (green) and eIF2a (light green). Initiation factor eIF1A (PDB: 2OQK) has been superpositioned to the bacterial 30S:IF1 complex (PDB: 1HR0), the eIF2: tRNAiMet ternary complex (PDB: 3V11) was aligned according to the P-site tRNA position in the bacterial 70S ribosome (PDB: 2WDK). The position of mRNA is also based on the 70S structure. Since atomic structures of eIF3 are not available, it is represented by an outline based on the superposition of eIF3:IRES and IRES:40S EM reconstructions [31]. (b) Interactions with eIF5 mapped on the 43S model. Regions of eIF1 (dark blue) that interact with eIF5 are highlighted in orange [28]. Orange spheres represent interactions involving the N-terminal extension of eIF2b (dark green) [26], the N-terminus of eIF1 [28] and the C-terminus of eIF1A (yellow) [29], which are not included in the model. (c) Crystal structures of the N-terminal and C-terminal domains of eIF5 (PDBs: 2G2K, 2FUL) show arbitrary orientation (orange). Regions that interact with eIF1 are indicated in dark blue [28,32], regions that interact with eIF2b in dark green [26], and regions required for eIF2g binding are highlighted in light green [24].
N-terminus of eIF1A points toward helix 30 (h30) of the small subunit head rRNA and is in proximity of proteins rpS15, rpS18 and rpS20 (Figure 2a). NMR structures (PDB: 1D7Q) reveal that the CTT of eIF1A is highly flexible, and hydroxyl radical probing data suggest that both tails interact mainly with the18S rRNA of the small subunit head, as well as the h24 stemloop on the platform [17]. On the basis of biochemical and electron microscopy (EM) data, it has been suggested that eIF1 and eIF1A jointly stabilize an ‘open’ conformation of the 43S complex that is competent for TC binding and scanning [5] (Scheme 1 panel 2). However, the limited resolution of the reconstruction and the lack of a 40S atomic model prevented the assignment of factors in the EM density [18] and high resolution data on larger PICs will ultimately be required to determine the combined structural roles of eIF1 and eIF1A. Current Opinion in Structural Biology 2012, 22:1–10
The 43S complex The heterotrimeric GTPase eIF2 not only delivers initiator tRNA to the ribosome, but also plays a key role in start codon selection [19]. The active, GTP-bound form binds to initiator tRNA and the resulting TC is recruited to the 40S. The GTPase-activating protein eIF5 stimulates GTP hydrolysis by eIF2, while the release of Pi is prevented by eIF1 until it dissociates upon start codon recognition (also termed irreversible GTP hydrolysis) [3] (Scheme 1 panel 3). The inactive GDP-bound form of eIF2 then dissociates from the complex, probably in complex with eIF5 [4], which inhibits the release of GDP [20]. Phosphorylation of the eIF2a subunit is a key mechanism of translational control and inhibits reactivation of eIF2:GDP by the pentameric guanylate exchange factor eIF2B [19]. The large eIF2g subunit contains the nucleotide and tRNA binding sites and is structurally related to elongation factors. Therefore, it has been suggested to www.sciencedirect.com
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Structural insights into eukaryotic ribosomes and the initiation of translation Voigts-Hoffmann, Klinge and Ban 5
Scheme 1
TC tRNAiMet
(1)
head
beak
40S
elF4 mRNA
(2)
Pi
(3)
elF1
elF2 GTP
elF1
(4)
elF1A elF3
body
left foot
“open” 43S PIC
recycled 40S complex
(7)
mRNA
start codon recognition
mRNA recruitment, scanning
“closed” PIC
central protuberance P-stalk
(8) L1-stalk
L1-stalk
P-stalk
tRNA
Efl1-GTP Efl1-GDP SBDS SBDS
E P A tunnel exit
elF5B
? elF6
elF1
recycling
elF6 elF3
elF2 GDP
recycled 60S
(180º rotated view) elF3
(6)
elF1A
(5)
elF4 complex
elongation termination
cycle elF1A
elF5B-GDP
80S IC
Translation initiation in eukaryotes. (1) A complex of 40S subunit and initiation factors eIF1 and eIF1A is joined by a ternary complex (TC) of initiator tRNAiMet and initiation factor eIF2-GTP to form the 43S pre-initiation complex (PIC). (2) Unwinding (‘‘activation’’) of mRNA and recruitment of the 43S PIC is catalyzed by eIF4, a large complex of several factors that mediate interactions with mRNA and the ribosome (Table 1). (3) The initiation complex scans the mRNA until an initiation codon in the appropriate context is encountered. Hydrolysis of eIF2-bound GTP is triggered by the GTPaseactivating protein (GAP) eIF5 on the ribosome, but inorganic phosphate (Pi) is not released until start codon recognition and dissociation of eIF1. It remains to be determined when and where eIF5 joins the initiation complex. (4) The initiation complex switches from an ‘‘open’’ (scanning competent), to a ‘‘closed’’ (arrested) state. Following the release of eIF2 and eIF5, subunit joining is mediated by eIF5B. (5) Hydrolysis of GTP by eIF5B is required for dissociation of eIF1A and eIF5B from the complex. (6) In the 80S initiation complex (IC), initiator tRNAiMet is basepaired to an AUG start codon in the ribosomal P-site and the A-site is empty. The ribosome is primed to enter the elongation cycle. (7) When the ribosome encounters a stop codon, the nascent polypeptide chain is detached by release factors. Recycling and initiation factors then effect the dissociation of subunits, mRNA and tRNA. (8) In panel 8, the 60S is shown from the interface side to illustrate the tRNA binding sites and the position of eIF6. The anti-association factor eIF6 is required during ribosome biogenesis and nuclear export, and the GTPase Efl1 and the SBDS protein in the cytoplasm catalyze its release from the large subunit. As explained in the text, several lines of evidence indicate that eIF6 also plays an important role in the regulation of translation.
bind to tRNA akin to elongation factor Tu (EF-Tu) [19]. However, the recent crystal structure of the archaeal TC (PDB: 3V11) reveals a different mode of binding, which involves the eIF2g and a subunits [8] (Figure 1c,e). Modeling the position of the TC on the 40S subunit (PDB: 2XZM) according to the P-site tRNA position in the bacterial ribosome orients domain III of eIF2g toward h44 of the 18S rRNA (Figure 2a). This is consistent with www.sciencedirect.com
hydroxyl radical probing of eIF2 interactions with tRNA and the ribosome [21]. The eIF2b core domain is mobile with respect to the eIF2g subunit and could not be unambiguously positioned in the crystallographic study [8]. Superposition of a heterotrimeric aIF2 structure determined in isolation (PDB: 2QMU) to the TC tentatively places the b subunit in proximity of h44, eIF1 and eIF1A (Figure 2a,b). Compared to the archaeal Current Opinion in Structural Biology 2012, 22:1–10
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6 Proteins
model, eukaryotic initiation factor 2 contains additional domains which may influence the mode of interaction with initiator tRNA [8,19,21,22]. Currently it is neither clear at which step during initiation eIF5 joins the 43S complex nor where it binds. However, several interactions between eIF5 and other initiation factors have been identified (Figure 2b,c). A partially disordered region of the eIF5 N-terminal domain (PDB: 2G2K) contains the arginine finger motif required for activation of the GTPase eIF2 [23,24]. The C-terminal HEAT domain of eIF5 (PDB: 2FUL) [25] is required for the inhibition of guanosine nucleotide dissociation from eIF2 [20] and its interactions with eIF2b are involved in the recruitment of eIF5 [26]. Moreover, eIF5 stimulates the dissociation of eIF1 [27] and its Cterminal domain interacts with the NTT and the second a helix of eIF1 [28]. eIF5 also increases the stability of eIF1A binding and reduces the mobility of its extreme Cterminus, but it is not clear whether this results from a direct interaction [29] (Figure 2b,c). Eukaryotic initiation factor 3 is a central organizing component of the 43S PIC and promotes TC binding, attachment of the 43S complex to mRNA and scanning. In mammals, eIF3 is a complex of 13 components, with a combined molecular weight exceeding 650 kDa. Five of these subunits are universally conserved, and the yeast complex includes orthologs of eIF3a, eIF3b, eIF3c, eIF3g and eIF3i [30] (Table 1). Although various interactions between eIF3 subunits and other initiation factors have been identified, structural information is limited, and atomic models are available only for selected domains of individual proteins. EM reconstructions reveal that eIF3 adopts a five-lobed overall shape and interacts with small subunit at the solventexposed side [31] (Scheme 1 and Figure 2). While this position would allow eIF3 to interact with mRNA and regulatory factors, higher resolution studies are required to localize the individual components and to understand their interactions in the 43S context.
is formed by the N-terminal G-domain, an EF-Tu-type b barrel and an aba sandwich. A helical linker connects the core domains to a second EF-Tu-type b barrel at the Cterminus (domain IV) and amplifies the modest structural rearrangements upon nucleotide exchange [38]. The structural homology of the eIF5B G-domain to EF-Tu and EF-G suggests a related mode of interaction with the ribosome [38], including the mechanism of GTPase activation. The crystal structure of eIF5B has been used to model IF2 in EM reconstructions of bacterial initiation complexes [39,40,41,42] but the conformational changes are larger than anticipated based on the crystal structures. In the 70S:IF1:IF2:IF3:tRNA complex (PDB: 1ZO1), the G-domain is located near the Sarcin–Ricin-Loop, in a position similar to EF-Tu (PDB: 2WRN), while the Cterminal b barrel contacts the aminoacceptor end of initiator tRNA in the ribosomal P-site [39] (Figure 3). This orientation corresponds to the GTP-bound form and agrees well with hydroxyl radical probing data of eIF5B on the 80S ribosome obtained in presence of a nonhydrolysable GTP analog [43]. Compared to the canonical P-site tRNA conformation (PDB: 2WDK), the elbow of the initiator tRNA is rotated toward the small subunit head and the E-site (Figure 3a), and a very similar conformation is also observed in the 30S initiation complexes [41,42]. As established in a different EM study following GTP hydrolysis and Pi release, domain IV resides in the ribosomal A-site and the G-domain is shifted downward [40,41]. Taken together, these results suggest that eIF5B mediates subunit joining by providing additional contacts between the subunits and by positioning the tRNA. During subunit joining its G-domain is optimally positioned to interact with the SRL region of the large subunit and upon GTP hydrolysis the factor undergoes significant conformational changes and ultimately dissociates along with eIF1A [37] (Scheme 1 panel 5).
Initiation factor 6 Initiation factor 5B In contrast to bacteria, translation initiation in eukaryotes requires two GTPases, eIF2 and eIF5B, for delivery of initiator tRNA and subunit joining, respectively, [1,2]. While bacterial IF2 recruits initiator tRNA to the 30S subunit [33], its eukaryotic ortholog eIF5B mainly functions in subunit joining [34] (Scheme 1 panel 5). An interaction with the CTT of eIF1A is required for recruitment and activation of eIF5B [35,36] and GTP hydrolysis by eIF5B enables the release of eIF5B and eIF1A upon subunit joining [37]. The crystal structure of eIF5B has been determined in isolation. The core of eIF5B (PDB: 1G7R, 1G7S, 1G7T) Current Opinion in Structural Biology 2012, 22:1–10
Initiation factor 6 is shared by eukaryotes (eIF6, Tif6p in yeast) and archaea (aIF6). It binds to a conserved site on the large ribosomal subunit in proximity to the Sarcin– Ricin-Loop and sterically precludes joining of the small subunit [10,44,45] (Figures 1b and 3b). The partitioning of eIF6 between nucleus and cytoplasm is controlled by phosphorylation. A nuclear pool of eIF6 is required during ribosome biogenesis and eIF6 remains bound to the large subunit during nuclear export [46]. Release of eIF6 is catalyzed by the GTPase Efl1 and the SBDS protein (sdo1p in yeast) [47] (Scheme 1 panel 8). In analogy to other translational GTPases, Efl1 may be activated through interactions with the ribosome, which www.sciencedirect.com
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Structural insights into eukaryotic ribosomes and the initiation of translation Voigts-Hoffmann, Klinge and Ban 7
Figure 3
(b)
(a)
central protuberance
tRNAiMet
beak
elF5B domain 4 tRNAiMet
P-stalk
C
mRNA
62Å
SRL
elF5B domain 4
elF1A
helical linker elF5B domains 1-3
elF5B domains 1-3
h44 right foot
90º
90º
left foot Current Opinion in Structural Biology
Homology models of eIF5B on the small and large subunit. (a) Models of eIF5B (blue), eIF1A (yellow), mRNA (red) and initiator tRNA (violet) docked on the small ribosomal subunit (PDBs: 3U5B, 3U5C). The binding interface of eIF1A and eIF5 is indicated by spheres and the yellow circle shows the region accessible to the flexible C-terminal tail of eIF1A in the most extended conformation (62 A˚) according to the NMR structure (PDB: 1D7Q). The distance between the truncated C-terminus of eIF1A and the residue with the strongest NMR interaction corresponds to (48 A˚) [36]. Initiation factor eIF1A (PDB: 2OQK) has been positioned on the small subunit according to the bacterial ortholog IF1 (PDB: 1HR0). The N-terminal and C-terminal regions of eIF5B (PDB: 1G7R) were individually positioned on the small subunit according to the EM model of the bacterial ortholog IF2 (PDB: 1ZO1). The position of initiator tRNA is based on the bacterial initiation complex. (PDB: 1ZO1). Ribosomal proteins are represented as light blue surfaces, ribosomal RNA as gray and white spheres. (b) Models of eIF5B and initiator tRNA on the large ribosomal subunit (PDBs: 3U5D, 3U5E) derived as described above. The relative position of the large subunit is based on the 80S ribosome (PDBs: 3U5B–3U5E).
may lead to conformational rearrangement that triggers the dissociation of eIF6 via SBDS [44,47]. Cytoplasmic eIF6 is required for the stimulation of translation in response to growth factor signaling in mammals [48], possibly because it prevents the formation of nontranslating 80S and regulates translation through 60S availability [6]. However, the mechanism by which the anti-association activity of eIF6 is regulated remains to be established.
Discussion The recent crystal structures of the 40S:eIF1 (PDB: 2XZM) and 60S:eIF6 (PDBs: 4A17, 4A19) complexes, eIF2-bound initiator tRNA (PDB: 3V11) and the 80S ribosome (PDBs: 3U5B, 3U5C), represent a significant progress toward a more complete understanding of translation in eukaryotes [7,8,9,10]. Compared to bacterial and archaeal ribosomes, eukaryotic ribosomes contain many additional proteins and rRNA elements, which form a network of interactions. With the availability of atomic structures, the functional roles of these www.sciencedirect.com
additional elements may now be scrutinized, using biochemical and genetic techniques (Figure 1). Combined with the structural data on bacterial initiation complexes and the structures of individual initiation factors or subcomplexes (Tables 1 and 2), initial models of the eukaryotic initiation complexes are beginning to emerge (Figures 2 and 3). Nevertheless, central mechanistic questions remain. For instance, it has not been determined how eIF1 and eIF5 control the GTPase activity and the release of Pi from eIF2, which commits the ribosome to initiation at a particular start codon. Initiation factors eIF1, eIF2b and the N-terminal domain of eIF5 are structurally related, sharing similar a/b folds [23]. This structural similarity may have several implications for the mechanism of start codon selection, including GTPase activation, Pi release and dissociation of eIF2 [5,19,23]. For instance, the displacement of eIF2b from the eIF2g subunit by the structurally related eIF5 NTD may stimulate the hydrolysis of eFI2g-bound GTP. Upon start codon recognition, eIF1 dissociates, which may allow the eIF5 NTD Current Opinion in Structural Biology 2012, 22:1–10
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8 Proteins
Table 2 List of structures. Experimental details and accession codes of structures discussed in the text or used for figure generation as described in the respective figure legend. A complete pymol session including the coordinate files of 40S, 60S and initiation factors as well as a pymol script for the superpositions is available for download on http://www.mol.biol.ethz.ch/groups/ban_group Structure
Source
Method
Res. [A˚]
40S:eIF1 40S:eIF1:eIF1A 60S:eIF6 80S:Stm1 30S:IF1 70S:tRNAs 70S:EF-Tu:tRNA 70S:IF1:IF2:IF3:mRNA:tRNA 30S:IF1:IF2:mRNA:tRNA 30S:IF1:IF2:IF3:mRNA:tRNA eIF1A eIF1A aIF5B aIF2:GDPNP:tRNA aIF2 (a, b, g)
euk euk euk euk bact bact bact bact bact bact euk euk arch arch arch
X-ray Cryo-EM X-ray X-ray X-ray X-ray X-ray EM EM EM X-ray NMR X-ray X-ray X-ray
3.9 22–25 3.5 3.0 3.2 3.5 3.6 13.8 8.7 18.3 1.8 n/a 2.0 5.0 3.2
Accession codes PDB: 2XZM EMD-1346, EMD-1347, EMD-1348, EMD-1349 PDB: 4A17, PDB: 4A19 PDB: 3U5B, PDB: 3U5C, PDB: 3U5D, PDB: 3U5E PDB: 1HR0 PDB: 2WDK, PDB: 2WDL PDB: 2WRN, PDB: 2WRO PDB: 1ZO1, PDB: 1ZO3, EMD-1248, EMD-1249 EMD-1523 EMD-1771 PDB: 2OQK PDB: 1D7Q PDB: 1G7R PDB: 3V11 PDB: 2QMU
n/a: not applicable.
to dissociate from eIF2g and bind to the 40S site previously occupied by eIF1, enabling Pi release from eFI2g. Whereas eIF5 does stimulate the dissociation of eIF1 upon start codon recognition there is no evidence for direct binding of eIF5 to the 40S and, therefore, the mechanistic relevance of the structural similarity between the factors remains to be established [5,19]. Further important aspects of start codon selection include the structural roles of eIF1 and eIF1A including its NTT and CTT. Crystal structures of eIF5B, the GTPase that mediates subunit joining, reveal modest rearrangements depending on the nucleotide binding state, which are amplified by a long helical linker between the structural core and the tRNA binding domain [38]. While the structural integrity of this helical linker indeed affects initiator tRNA binding and subunit joining [49], the crystal structures do not fully account for the domain arrangement in EM reconstructions of ribosomal IF2 complexes corresponding to the state before GTP hydrolysis [38– 42]. Small angle X-ray scattering studies suggest that contacts with initiator tRNA and the ribosome are required for the GTPase to assume its active conformation [50]. The models of eukaryotic initiation complexes described herein are based on the recently determined crystal structures of the eukaryotic ribosome, cryo-EM reconstructions of bacterial initiation complexes and structures of individual initiation factors. They provide first insights into how larger PICs may be organized. While these data may guide the design of biochemical, genetic and spectroscopic experiments, further structures of initiation complexes trapped at different stages of the process will Current Opinion in Structural Biology 2012, 22:1–10
ultimately be required to better understand the molecular mechanisms that govern eukaryotic translation initiation.
Acknowledgements We thank Daniel Bo¨hringer for the superposition of EM structures and Jan Erzberger for his support in drafting the article and critical reading of the manuscript.
Appendix A. Supplementary data Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/ j.sbi.2012.07.010.
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest 1.
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2.
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Singh CR, Lee B, Udagawa T, Mohammad-Qureshi SS, Yamamoto Y, Pavitt GD, Asano K: An eIF5/eIF2 complex antagonizes guanine nucleotide exchange by eIF2B during translation initiation. EMBO J 2006, 25:4537-4546.
5.
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6.
Brina D, Grosso S, Miluzio A, Biffo S: Translational control by 80S formation and 60S availability: the central role of eIF6, a rate limiting factor in cell cycle progression and tumorigenesis. Cell Cycle 2011, 10:3441-3446. www.sciencedirect.com
Please cite this article in press as: Voigts-Hoffmann F, et al.: Structural insights into eukaryotic ribosomes and the initiation of translation, Curr Opin Struct Biol (2012), http://dx.doi.org/10.1016/ j.sbi.2012.07.010
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Structural insights into eukaryotic ribosomes and the initiation of translation Voigts-Hoffmann, Klinge and Ban 9
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Rabl J, Leibundgut M, Ataide SF, Haag A, Ban N: Crystal structure of the eukaryotic 40S ribosomal subunit in complex with initiation factor 1. Science 2011, 331:730-736. This paper describes the structure of the eukaryotic small ribosomal subunit in complex with initiation factor 1, a key regulator in start codon selection. It is the first atomic structure of a eukaryotic initiation complex.
8.
22. Naveau M, Lazennec-Schurdevin C, Panvert M, Mechulam Y, Schmitt E: tRNA binding properties of eukaryotic translation initiation factor 2 from Encephalitozoon cuniculi. Biochemistry 2010, 49:8680-8688. 23. Conte MR, Kelly G, Babon J, Sanfelice D, Youell J, Smerdon SJ, Proud CG: Structure of the eukaryotic initiation factor (eIF) 5 reveals a fold common to several translation factors. Biochemistry 2006, 45:4550-4558.
Schmitt E, Panvert M, Lazennec-Schurdevin C, Coureux P-D, Perez J, Thompson A, Mechulam Y: Structure of the ternary initiation complex aIF2-GDPNP-methionylated initiator tRNA. Nat Struct Mol Biol 2012, 19:450-454. The structure of the ternary complex reveals that the tRNA binding mode of eIF2 differs significantly from EF-Tu, which is in agreement with earlier biochemical investigations [21].
24. Alone PV: Direct binding of translation initiation factor eIF2g-G domain to its GTPase-activating and GDP–GTP exchange factors eIF5 and eIF2B epsilon. J Biol Chem 2006, 281:1263612644.
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25. Wei Z, Xue Y, Xu H, Gong W: Crystal structure of the C-terminal domain of S. cerevisiae eIF5. J Mol Biol 2006, 359:1-9.
Ben-Shem A, Garreau de Loubresse N, Melnikov S, Jenner L, Yusupova G, Yusupov M: The structure of the eukaryotic ribosome at 3.0 A resolution. Science 2011, 334:1524-1529. The structure of the 80S from yeast ribosome is the highest resolved eukaryotic ribosome structure and describes additional contacts involving segments that are disordered in the individual subunits. 10. Klinge S, Voigts-Hoffmann F, Leibundgut M, Arpagaus S, Ban N: Crystal structure of the eukaryotic 60S ribosomal subunit in complex with initiation factor 6. Science 2011, 334:941-948. This complex is the first complete, atomic model of the eukaryotic large ribosomal subunit and reveals the details of the interaction with initiation factor 6.
11. Klinge S, Voigts-Hoffmann F, Leibundgut M: Atomic structures of the eukaryotic ribosome. Trends Biochem Sci 2012:37. Comprehensive discussion of the architectural concepts that emerge from the recent crystal structures of the eukaryotic ribosome. 12. Jackson RJ, Hellen CUT, Pestova TV: Termination and posttermination events in eukaryotic translation. Adv Protein Chem Struct Biol 2012, 86:45-93. 13. Lomakin IB, Kolupaeva VG, Marintchev A, Wagner G, Pestova TV: Position of eukaryotic initiation factor eIF1 on the 40S ribosomal subunit determined by directed hydroxyl radical probing. Genes Dev 2003, 17:2786-2797. 14. Martin-Marcos P, Cheung Y-N, Hinnebusch AG: Functional elements in initiation factors 1, 1A, and 2b discriminate against poor AUG context and non-AUG start codons. Mol Cell Biol 2011, 31:4814-4831.
26. Asano K, Krishnamoorthy T, Phan L, Pavitt GD, Hinnebusch AG: Conserved bipartite motifs in yeast eIF5 and eIF2Bepsilon, GTPase-activating and GDP–GTP exchange factors in translation initiation, mediate binding to their common substrate eIF2. EMBO J 1999, 18:1673-1688. 27. Nanda JS, Cheung Y-N, Takacs JE, Martin-Marcos P, Saini AK, Hinnebusch AG, Lorsch JR: eIF1 controls multiple steps in start codon recognition during eukaryotic translation initiation. J Mol Biol 2009, 394:268-285. 28. Reibarkh M, Yamamoto Y, Singh CR, del Rio F, Fahmy A, Lee B, Luna RE, Ii M, Wagner G, Asano K: Eukaryotic initiation factor (eIF) 1 carries two distinct eIF5-binding faces important for multifactor assembly and AUG selection. J Biol Chem 2008, 283:1094-1103. 29. Maag D, Algire MA, Lorsch JR: Communication between eukaryotic translation initiation factors 5 and 1A within the ribosomal pre-initiation complex plays a role in start site selection. J Mol Biol 2006, 356:724-737. 30. Hinnebusch AG: eIF3: a versatile scaffold for translation initiation complexes. Trends Biochem Sci 2006, 31:553-562. 31. Siridechadilok B, Fraser CS, Hall RJ, Doudna JA, Nogales E: Structural roles for human translation factor eIF3 in initiation of protein synthesis. Science 2005, 310:1513-1515.
15. Fekete CA, Mitchell SF, Cherkasova VA, Applefield D, Algire MA, Maag D, Saini AK, Lorsch JR, Hinnebusch AG: N- and C-terminal residues of eIF1A have opposing effects on the fidelity of start codon selection. EMBO J 2007, 26:1602-1614.
32. Yamamoto Y, Singh CR, Marintchev A, Hall NS, Hannig EM, Wagner G, Asano K: The eukaryotic initiation factor (eIF) 5 HEAT domain mediates multifactor assembly and scanning with distinct interfaces to eIF1, eIF2, eIF3, and eIF4G. Proc Natl Acad Sci U S A 2005, 102:16164-16169.
16. Saini AK, Nanda JS, Lorsch JR, Hinnebusch AG: Regulatory elements in eIF1A control the fidelity of start codon selection by modulating tRNA(i)(Met) binding to the ribosome. Genes Dev 2010, 24:97-110.
33. Milon P, Carotti M, Konevega AL, Wintermeyer W, Rodnina MV, Gualerzi CO: The ribosome-bound initiation factor 2 recruits initiator tRNA to the 30S initiation complex. EMBO Rep 2010, 11:312-316.
17. Yu Y, Marintchev A, Kolupaeva VG, Unbehaun A, Veryasova T, Lai S-C, Hong P, Wagner G, Hellen CUT, Pestova TV: Position of eukaryotic translation initiation factor eIF1A on the 40S ribosomal subunit mapped by directed hydroxyl radical probing. Nucleic Acids Res 2009, 37:5167-5182.
34. Pestova TV, Lomakin IB, Lee JH, Choi SK, Dever TE, Hellen CU: The joining of ribosomal subunits in eukaryotes requires eIF5B. Nature 2000, 403:332-335.
18. Passmore LA, Schmeing TM, Maag D, Applefield DJ, Acker MG, Algire MA, Lorsch JR, Ramakrishnan V: The eukaryotic translation initiation factors eIF1 and eIF1A induce an open conformation of the 40S ribosome. Mol Cell 2007, 26:41-50. 19. Schmitt E, Naveau M, Mechulam Y: Eukaryotic and archaeal translation initiation factor 2: a heterotrimeric tRNA carrier. FEBS Lett 2010, 584:405-412. 20. Jennings MD, Pavitt GD: eIF5 has GDI activity necessary for translational control by eIF2 phosphorylation. Nature 2010, 465:378-381. 21. Shin B-S, Kim J-R, Walker SE, Dong J, Lorsch JR, Dever TE: Initiation factor eIF2g promotes eIF2–GTP–Met-tRNAi(Met) ternary complex binding to the 40S ribosome. Nat Struct Mol Biol 2011, 18:1227-1234. By careful biochemical investigation, the authors disprove popular assumptions regarding the structure of ternary complex and derive a new model, which is substantiated by later crystallographic studies [8]. Furthermore, the authors examine the interactions between ternary complex and the ribosome. www.sciencedirect.com
35. Acker MG, Shin B-S, Dever TE, Lorsch JR: Interaction between eukaryotic initiation factors 1A and 5B is required for efficient ribosomal subunit joining. J Biol Chem 2006, 281:8469-8475. 36. Marintchev A, Kolupaeva VG, Pestova TV, Wagner G: Mapping the binding interface between human eukaryotic initiation factors 1A and 5B: a new interaction between old partners. Proc Natl Acad Sci U S A 2003, 100:1535-1540. 37. Fringer JM, Acker MG, Fekete CA, Lorsch JR, Dever TE: Coupled release of eukaryotic translation initiation factors 5B and 1A from 80S ribosomes following subunit joining. Mol Cell Biol 2007, 27:2384-2397. 38. Roll-Mecak A, Cao C, Dever TE, Burley SK: X-ray structures of the universal translation initiation factor IF2/eIF5B: conformational changes on GDP and GTP binding. Cell 2000, 103:781-792. 39. Allen GS, Zavialov A, Gursky R, Ehrenberg M, Frank J: The cryo EM structure of a translation initiation complex from Escherichia coli. Cell 2005, 121:703-712. EM structure of the bacterial initiation complex, based on the crystal structure of archaeal eIF5B. This structure also provides the basis for the homology models in Figure 3. Current Opinion in Structural Biology 2012, 22:1–10
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40. Myasnikov AG, Marzi S, Simonetti A, Giuliodori AM, Gualerzi CO, Yusupova G, Yusupov M, Klaholz BP: Conformational transition of initiation factor 2 from the GTP- to GDP-bound state visualized on the ribosome. Nat Struct Mol Biol 2005, 12:1145-1149. 41. Simonetti A, Marzi S, Myasnikov AG, Fabbretti A, Yusupov M, Gualerzi CO, Klaholz BP: Structure of the 30S translation initiation complex. Nature 2008, 455:416-420. EM study of a small subunit bacterial initiation complex that illustrates the conformational rearrangements of eIF5B/IF2. 42. Julia´n P, Milon P, Agirrezabala X, Lasso G, Gil D, Rodnina MV, Valle M: The Cryo-EM structure of a complete 30S translation initiation complex from Escherichia coli. PLoS Biol 2011, 9:e1001095. 43. Unbehaun A, Marintchev A, Lomakin IB, Didenko T, Wagner G, Hellen CUT, Pestova TV: Position of eukaryotic initiation factor eIF5B on the 80S ribosome mapped by directed hydroxyl radical probing. EMBO J 2007, 26:3109-3123. 44. Gartmann M, Blau M, Armache JP, Mielke T, Topf M, Beckmann R: Mechanism of eIF6-mediated inhibition of ribosomal subunit joining. J Biol Chem 2010, 285:14848-14851. 45. Greber BJ, Boehringer D, Godinic-Mikulcic V, Crnkovic A, Ibba M, Weygand-Durasevic I, Ban N: Cryo-EM structure of the archaeal 50S ribosomal subunit in complex with initiation factor 6 and
Current Opinion in Structural Biology 2012, 22:1–10
implications for ribosome evolution. J Mol Biol 2012, 418:145-160. 46. Biswas A, Mukherjee S, Das S, Shields D, Chow CW, Maitra U: Opposing action of casein kinase 1 and calcineurin in nucleocytoplasmic shuttling of mammalian translation initiation factor eIF6. J Biol Chem 2011, 286:3129-3138. 47. Finch AJ, Hilcenko C, Basse N, Drynan LF, Goyenechea B, Menne TF, Gonzalez Fernandez A, Simpson P, D’Santos CS, Arends MJ et al.: Uncoupling of GTP hydrolysis from eIF6 release on the ribosome causes Shwachman–Diamond syndrome. Genes Dev 2011, 25:917-929. 48. Gandin V, Miluzio A, Barbieri AM, Beugnet A, Kiyokawa H, Marchisio PC, Biffo S: Eukaryotic initiation factor 6 is ratelimiting in translation, growth and transformation. Nature 2008, 455:684-688. 49. Shin BS, Acker MG, Kim JR, Maher KN, Arefin SM, Lorsch JR, Dever TE: Structural integrity of a-helix H12 in translation initiation factor eIF5B is critical for 80S complex stability. RNA 2011, 17:687-696. 50. Vohlander Rasmussen LC, Oliveira CLP, Pedersen JS, SperlingPetersen HU, Mortensen KK: Structural transitions of translation initiation factor IF2 upon GDPNP and GDP binding in solution. Biochemistry 2011, 50:9779-9787.
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Available online at www.sciencedirect.com
Structural insights into eukaryotic ribosomes and the initiation of translation§ Felix Voigts-Hoffmann, Sebastian Klinge and Nenad Ban The initiation of protein biosynthesis entails the ordered assembly of elongation-competent ribosomes, with an initiator tRNA basepaired to an appropriate mRNA start codon. In eukaryotes, this process is more complex than in prokaryotes and involves numerous protein factors that mediate tRNA delivery, mRNA binding, start codon selection and subunit joining. The recent 40S:eIF1, 80S and eIF2:tRNA:GDPNP ternary complex structures provide an initial structural framework toward a molecular understanding of the eukaryotic translation initiation process. Updated homology models of larger initiation complexes provide first insights into the likely arrangements of these higher-order complexes, but also reveal the limits of our current understanding of the eukaryotic translation initiation process. Address Institute of Molecular Biology and Biophysics, ETH Zurich, 8093 Zurich, Switzerland Corresponding author: Ban, Nenad ([email protected])
Current Opinion in Structural Biology 2012, 22:xx–yy This review comes from a themed issue on Proteins Edited by Anders Liljas and Peter Moore
0959-440X/$ – see front matter, # 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.sbi.2012.07.010
Introduction Translation initiation refers to the formation of translation-competent ribosomes with initiator tRNA basepaired to the mRNA start codon at the ribosomal Psite. Bacteria rely on the Shine–Dalgarno sequence of the mRNA to locate the start codon and require just three initiation factors [1]. In contrast, eukaryotic translation initiation relies on a scanning mechanism to locate the start codon and involves 13 core initiation factors, some of which are large, multimeric complexes [2] (Table 1).
§ Funding sources: We acknowledge support by the Swiss National Science Foundation (SNSF), the National Center of Excellence in Research (NCCR) Structural Biology program of the SNSF, and European Research Council grant 250071 under the European Community’s Seventh Framework Programme. S.K. was supported by EMBO and HFSP Fellowships.
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In eukaryotes, initiator tRNA is delivered to the ribosome as a ternary complex (TC) with eIF2 and GTP (Scheme 1 panel 1). Initiation factors eIF1, eIF1A and eIF3 promote the binding of the TC to form a 43S pre-initiation complex (PIC). The 43S PIC binds to the mRNA through its interactions with the mRNA cap-binding complex eIF4 and begins scanning for the start codon (Scheme 1 panel 2). Hydrolysis of eIF2-bound GTP is the key step in translation initiation, which commits the 43S complex to initiation at a particular start codon. It is controlled by eIF5, which stimulates the hydrolysis of eIF2-bound GTP within the initiation complex, and eIF1, which blocks the release of inorganic phosphate (Pi) [3]. (Scheme 1 panel 3) Upon start codon recognition, eIF1 is displaced from the P-site, leading to the release of Pi and dissociation of eIF2, probably in complex with eIF5 [4] (Scheme 1 panel 4). A second GTPase, eIF5B, then mediates subunit joining and displacement of initiation factors. (Scheme 1 panel 5) Hydrolysis of eIF5B-bound GTP is required for dissociation of eIF5B and eIF1A, vacating the A-site for the first elongator tRNA (Scheme 1 panel 6, for details refer to reviews [1,2,5]). The initiation process is regulated primarily at the level of TC formation and mRNA recruitment [1,2,5], and may be modulated through the availability of the large ribosomal subunit [6]. However, the molecular mechanisms of eukaryotic translation initiation remain to be established. The crystal structures of the 40S:eIF1 complex [7] (PDB: 2XZM), the TC [8] (PDB: 3V11) and the 80S ribosome [9] (PDBs: 3U5B–3U5E) provide an opportunity to generate models of initiation intermediates as a starting point for further analysis (Figure 1).
Structures of the eukaryotic ribosome Compared to prokaryotes, the increased complexity of eukaryotic protein synthesis is not only reflected in the initiation process, but also in the architecture of the ribosome itself [1,2,5,7,9,10]. Whereas the core functional regions such as the peptidyl transferase center (PTC) or the decoding site are conserved between domains of life, eukaryotic ribosomes contain many additional rRNA and protein segments, and some regions, such as the beak and the left foot of the small subunit, have been extensively remodeled. Furthermore, eukaryote-specific protein segments engage in extensive protein–protein contacts, while prokaryotic ribosomal Current Opinion in Structural Biology 2012, 22:1–10
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2 Proteins
Table 1 Eukaryotic core initiation factors. Molecular weights were calculated based on the human amino acid sequence Initiation factor
Bacterial homolog
Subunits
eIF1
None
1 (12.7 kDa)
eIF1A
IF1
1 (16.5 kDa)
eIF2
None
eIF2B
None
eIF3
None
a (36.1 kDa) b (38.4 kDa) g (51.1 kDa) a (33.7 kDa) b (39 kDa) g (50.2 kDa) d (57.6 kDa) e (80.4 kDa) a–ma (790 kDa)
eIF4A eIF4B eIF4E eIF4F
None None None None
1 (46 kDa) 1 (69.1 kDa) 1 (25.1 kDa) E, A, G (140.2)
eIF4G
None
1 (175.5 kDa)
eIF4H eIF5 eIF5B
None None IF2
1 (27.4 kDa) 1 (49.2 kDa) 1 (138.8 kDa)
Function Prevents initiation at noncognate codons or in poor context. Functionally related to bacterial IF3 Cooperates with eIF1 in start codon selection, and is involved in the recruitment of eIF5B GTPase that forms a ternary complex (TC) with initiator tRNA and GTP. Plays a key role in start codon selection Nucleotide exchange factor required for re-activation of eIF2
Stimulates binding of ternary complex (TC) and attachment of 43S complexes to mRNA and participates in scanning DEAD-box ATPase and ATP-dependent RNA helicase Enhances activity of eIF4A Binds to the mRNA cap structure Synonym for a complex of eIF4E, eIF4A and eIF4G. Mediates unwinding of mRNA and attachment of 43S complexes Binds to eIF4E, eIF4A, eIF3, PABP, SLIP1 and mRNA and enhances the helicase activity of elF4A Enhances activity of eIF4A, partially homologous to eIF4B GAP and GDI regulating eIF2 activity GTPase that mediates subunit joining
Adapted from [2]. a
eIF3 has 13 subunits (a–m) in mammals, 5 of which (a, b, c, i, g) are universally conserved.
proteins interact mainly with rRNA. This interconnectivity is particularly evident on the solvent-exposed side of the large subunit, where extensions of ribosomal proteins and novel proteins form an intricate network of interactions with the rRNA expansion segments (for a detailed discussion of the eukaryotic ribosome structures, refer to [7,9,10,11]). While some of the eukaryote-specific proteins may function to stabilize the structure of the ribosomal rRNA [10], others may participate directly in the regulation of translation. For instance, small subunit protein rpS6 is phosphorylated downstream of the mammalian target of rapamycin (mTOR) kinase, which integrates stimuli such as growth factor signaling [7]. With the availability of atomic models [7,9,10], the various novel components of the eukaryotic ribosome may now be characterized to determine structural and functional roles (Figure 1).
Binding of eIF1 and eIF1A to the small subunit Together with eIF2, eIF1 and eIF1A are the main determinants of start codon selection. The presence of eIF1 prevents initiation on codons that are noncognate or present in an improper context, such as a short leader sequence. eIF1 also blocks the release of Pi from eIF2– GDP–Pi in the 43S PIC [3] (Scheme 1 panel 2). The eukaryotic ortholog of bacterial IF1, eIF1A, cooperates with eIF1 in start codon selection and is also involved in the recruitment of eIF5B, the GTPase that mediates Current Opinion in Structural Biology 2012, 22:1–10
subunit joining [5]. Furthermore, eIF1 and eIF1A participate in ribosome recycling: Binding of eIF1, eIF1A and eIF3 to recycled 40S subunits leads to the release of deacetylated P-site tRNA and dissociation of mRNA, while the factors remain bound to the 40S, priming it for the next round of initiation [12] (Scheme 1 panel 7). The crystal structure of the eukaryotic 40S subunit in complex with eIF1 (PDB: 2XZM) reveals that eIF1 binds to the interface side of the 40S body, in proximity to the P-site [7] (Figure 1). eIF1 binds to helix 44 (h44) of the small subunit 18S rRNA via a positively charged surface region. Furthermore, several residues of the basic loop (residues 26–30 in Tetrahymena thermophila) are positioned in close proximity to the mRNA channel. In contrast, the region facing toward the P-site tRNA contains acidic and hydrophobic residues. Steric clashes between eIF1 and tRNA in the canonical P-site orientation (PDBs: 2WDL, 2WDK) suggest that the factor may be displaced by initiator tRNA upon formation of cognate start codon interactions. In support of this model, hydroxyl radical probing data indicate that initiator tRNA may be slightly rotated in the 40S:eIF1 complex [13] (Scheme 1 panel 4, Figure 2). Steric hindrance also underlies the anti-association activity of eIF1. Because its binding site overlaps with the position of the highly conserved helix 69 (h69) of the large subunit 26S rRNA in the 80S ribosome, eIF1 will prevent joining of the large subunit to initiation complexes (Figure 1). www.sciencedirect.com
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Structural insights into eukaryotic ribosomes and the initiation of translation Voigts-Hoffmann, Klinge and Ban 3
Figure 1
(a)
central protuberance
(b) rpS19
rpS18 rpS15
RPL5
head
RPL11
rpS25 rpS5 rpS16
beak
RPL36A (L42) RPL13
rpS28
rpS27A (S31)
RPL10
RPLP0
h69 platform
elF1
P-stalk
RPL4
RPL36
rpS14
RPL7A (L8)
shoulder
RPL40
rpS30
rpS3A (S1)
rpS23
RPL9
RPL8 (L2)
RPL23
RPL37A (L43)
rpS24
RPL34
rpS27
rpS6
elF6
rpS13 RPL27 right foot
rpS11
RPL3
RPL30 RPL38
body
rpS8
RPL24 RPL19
RPL22
left foot
(c)
(d) elF2γ
beak
head P-stalk
elF2α
elF2β RACK1
tRNAiMet
40S
60S
EF-Tu body tRNA
Current Opinion in Structural Biology
(a) Crystal structures of the eukaryotic 40S:eIF1 complex (PDB: 2XZM) and (b) the 60S:eIF6 complex (PDBs: 4A17, 4A19) from Tetrahymena thermophila. Ribosomal rRNA is depicted in gray, ribosomal proteins in different colors. Proteins are labeled according to UNIPROT nomenclature, and yeast protein names are indicated in parentheses, where different. (c) Crystal structure of archaeal (Sulfolobussolfataricus) ternary initiation complex aIF2:GDPNP:initiator-tRNA (PDB: 3V11) compared to bacterial (Escherichia coli) elongation factor EF-Tu:GTP:tRNA ternary complex (PDB: 1B23). (d) Crystal structure of the Saccharomyces cerevisiae 80S ribosome (PDBs: 3U5B–3U5E). The 40S subunit in (a) is rotated 1108 clockwise relative to the position in the 80S (d), the 60S subunit in (b) 708 counterclockwise (as seen from the top).
Modeling of eIF1A (PDB: 2OQK) on the 40S based on the bacterial 30S:IF1 complex (PDB: 1HR0) positions it in the A-site, in contact with h44 and h18 of the 18S rRNA and ribosomal proteins rpS23, rpS27A and rpS30 (Figure 2a). Compared to bacterial IF1, eIF1A has an www.sciencedirect.com
additional alpha helix as well as N-terminal tail (NTT) and C-terminal tails (CTT) that inhibit and enhance scanning, respectively [14–16]. In the superposition, the truncated C-terminus of the eIF1A structure is oriented toward the P-site and eIF1, whereas the Current Opinion in Structural Biology 2012, 22:1–10
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4 Proteins
Figure 2
(a)
N
(b)
mRNA C
elF3
elF2α
N
beak
elF1A
elF1 C
elF2β
tRNAiMet
C
elF2α
elF1A
N elF2γ N
C
(c)
elF2β elF2γ
156
elF5 CTD
N 237 elF2β right foot
left foot
elF5 NTD
C elF1
elF2γ Current Opinion in Structural Biology
(a) Homology model of a small subunit pre-initiation complex. The model is based on the crystal structure of the 40S:eIF1 complex (PDB: 2XZM). 40S ribosomal RNA is shown as gray and white spheres, proteins as light blue surfaces. Initiation factors, mRNA and tRNA are shown as cartoons with transparent surface and colored as follows: P-site tRNA (violet), eIF1 (dark blue), eIF1A (yellow), mRNA (red), eIF2b (dark green), eIF2g (green) and eIF2a (light green). Initiation factor eIF1A (PDB: 2OQK) has been superpositioned to the bacterial 30S:IF1 complex (PDB: 1HR0), the eIF2: tRNAiMet ternary complex (PDB: 3V11) was aligned according to the P-site tRNA position in the bacterial 70S ribosome (PDB: 2WDK). The position of mRNA is also based on the 70S structure. Since atomic structures of eIF3 are not available, it is represented by an outline based on the superposition of eIF3:IRES and IRES:40S EM reconstructions [31]. (b) Interactions with eIF5 mapped on the 43S model. Regions of eIF1 (dark blue) that interact with eIF5 are highlighted in orange [28]. Orange spheres represent interactions involving the N-terminal extension of eIF2b (dark green) [26], the N-terminus of eIF1 [28] and the C-terminus of eIF1A (yellow) [29], which are not included in the model. (c) Crystal structures of the N-terminal and C-terminal domains of eIF5 (PDBs: 2G2K, 2FUL) show arbitrary orientation (orange). Regions that interact with eIF1 are indicated in dark blue [28,32], regions that interact with eIF2b in dark green [26], and regions required for eIF2g binding are highlighted in light green [24].
N-terminus of eIF1A points toward helix 30 (h30) of the small subunit head rRNA and is in proximity of proteins rpS15, rpS18 and rpS20 (Figure 2a). NMR structures (PDB: 1D7Q) reveal that the CTT of eIF1A is highly flexible, and hydroxyl radical probing data suggest that both tails interact mainly with the18S rRNA of the small subunit head, as well as the h24 stemloop on the platform [17]. On the basis of biochemical and electron microscopy (EM) data, it has been suggested that eIF1 and eIF1A jointly stabilize an ‘open’ conformation of the 43S complex that is competent for TC binding and scanning [5] (Scheme 1 panel 2). However, the limited resolution of the reconstruction and the lack of a 40S atomic model prevented the assignment of factors in the EM density [18] and high resolution data on larger PICs will ultimately be required to determine the combined structural roles of eIF1 and eIF1A. Current Opinion in Structural Biology 2012, 22:1–10
The 43S complex The heterotrimeric GTPase eIF2 not only delivers initiator tRNA to the ribosome, but also plays a key role in start codon selection [19]. The active, GTP-bound form binds to initiator tRNA and the resulting TC is recruited to the 40S. The GTPase-activating protein eIF5 stimulates GTP hydrolysis by eIF2, while the release of Pi is prevented by eIF1 until it dissociates upon start codon recognition (also termed irreversible GTP hydrolysis) [3] (Scheme 1 panel 3). The inactive GDP-bound form of eIF2 then dissociates from the complex, probably in complex with eIF5 [4], which inhibits the release of GDP [20]. Phosphorylation of the eIF2a subunit is a key mechanism of translational control and inhibits reactivation of eIF2:GDP by the pentameric guanylate exchange factor eIF2B [19]. The large eIF2g subunit contains the nucleotide and tRNA binding sites and is structurally related to elongation factors. Therefore, it has been suggested to www.sciencedirect.com
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Structural insights into eukaryotic ribosomes and the initiation of translation Voigts-Hoffmann, Klinge and Ban 5
Scheme 1
TC tRNAiMet
(1)
head
beak
40S
elF4 mRNA
(2)
Pi
(3)
elF1
elF2 GTP
elF1
(4)
elF1A elF3
body
left foot
“open” 43S PIC
recycled 40S complex
(7)
mRNA
start codon recognition
mRNA recruitment, scanning
“closed” PIC
central protuberance P-stalk
(8) L1-stalk
L1-stalk
P-stalk
tRNA
Efl1-GTP Efl1-GDP SBDS SBDS
E P A tunnel exit
elF5B
? elF6
elF1
recycling
elF6 elF3
elF2 GDP
recycled 60S
(180º rotated view) elF3
(6)
elF1A
(5)
elF4 complex
elongation termination
cycle elF1A
elF5B-GDP
80S IC
Translation initiation in eukaryotes. (1) A complex of 40S subunit and initiation factors eIF1 and eIF1A is joined by a ternary complex (TC) of initiator tRNAiMet and initiation factor eIF2-GTP to form the 43S pre-initiation complex (PIC). (2) Unwinding (‘‘activation’’) of mRNA and recruitment of the 43S PIC is catalyzed by eIF4, a large complex of several factors that mediate interactions with mRNA and the ribosome (Table 1). (3) The initiation complex scans the mRNA until an initiation codon in the appropriate context is encountered. Hydrolysis of eIF2-bound GTP is triggered by the GTPaseactivating protein (GAP) eIF5 on the ribosome, but inorganic phosphate (Pi) is not released until start codon recognition and dissociation of eIF1. It remains to be determined when and where eIF5 joins the initiation complex. (4) The initiation complex switches from an ‘‘open’’ (scanning competent), to a ‘‘closed’’ (arrested) state. Following the release of eIF2 and eIF5, subunit joining is mediated by eIF5B. (5) Hydrolysis of GTP by eIF5B is required for dissociation of eIF1A and eIF5B from the complex. (6) In the 80S initiation complex (IC), initiator tRNAiMet is basepaired to an AUG start codon in the ribosomal P-site and the A-site is empty. The ribosome is primed to enter the elongation cycle. (7) When the ribosome encounters a stop codon, the nascent polypeptide chain is detached by release factors. Recycling and initiation factors then effect the dissociation of subunits, mRNA and tRNA. (8) In panel 8, the 60S is shown from the interface side to illustrate the tRNA binding sites and the position of eIF6. The anti-association factor eIF6 is required during ribosome biogenesis and nuclear export, and the GTPase Efl1 and the SBDS protein in the cytoplasm catalyze its release from the large subunit. As explained in the text, several lines of evidence indicate that eIF6 also plays an important role in the regulation of translation.
bind to tRNA akin to elongation factor Tu (EF-Tu) [19]. However, the recent crystal structure of the archaeal TC (PDB: 3V11) reveals a different mode of binding, which involves the eIF2g and a subunits [8] (Figure 1c,e). Modeling the position of the TC on the 40S subunit (PDB: 2XZM) according to the P-site tRNA position in the bacterial ribosome orients domain III of eIF2g toward h44 of the 18S rRNA (Figure 2a). This is consistent with www.sciencedirect.com
hydroxyl radical probing of eIF2 interactions with tRNA and the ribosome [21]. The eIF2b core domain is mobile with respect to the eIF2g subunit and could not be unambiguously positioned in the crystallographic study [8]. Superposition of a heterotrimeric aIF2 structure determined in isolation (PDB: 2QMU) to the TC tentatively places the b subunit in proximity of h44, eIF1 and eIF1A (Figure 2a,b). Compared to the archaeal Current Opinion in Structural Biology 2012, 22:1–10
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6 Proteins
model, eukaryotic initiation factor 2 contains additional domains which may influence the mode of interaction with initiator tRNA [8,19,21,22]. Currently it is neither clear at which step during initiation eIF5 joins the 43S complex nor where it binds. However, several interactions between eIF5 and other initiation factors have been identified (Figure 2b,c). A partially disordered region of the eIF5 N-terminal domain (PDB: 2G2K) contains the arginine finger motif required for activation of the GTPase eIF2 [23,24]. The C-terminal HEAT domain of eIF5 (PDB: 2FUL) [25] is required for the inhibition of guanosine nucleotide dissociation from eIF2 [20] and its interactions with eIF2b are involved in the recruitment of eIF5 [26]. Moreover, eIF5 stimulates the dissociation of eIF1 [27] and its Cterminal domain interacts with the NTT and the second a helix of eIF1 [28]. eIF5 also increases the stability of eIF1A binding and reduces the mobility of its extreme Cterminus, but it is not clear whether this results from a direct interaction [29] (Figure 2b,c). Eukaryotic initiation factor 3 is a central organizing component of the 43S PIC and promotes TC binding, attachment of the 43S complex to mRNA and scanning. In mammals, eIF3 is a complex of 13 components, with a combined molecular weight exceeding 650 kDa. Five of these subunits are universally conserved, and the yeast complex includes orthologs of eIF3a, eIF3b, eIF3c, eIF3g and eIF3i [30] (Table 1). Although various interactions between eIF3 subunits and other initiation factors have been identified, structural information is limited, and atomic models are available only for selected domains of individual proteins. EM reconstructions reveal that eIF3 adopts a five-lobed overall shape and interacts with small subunit at the solventexposed side [31] (Scheme 1 and Figure 2). While this position would allow eIF3 to interact with mRNA and regulatory factors, higher resolution studies are required to localize the individual components and to understand their interactions in the 43S context.
is formed by the N-terminal G-domain, an EF-Tu-type b barrel and an aba sandwich. A helical linker connects the core domains to a second EF-Tu-type b barrel at the Cterminus (domain IV) and amplifies the modest structural rearrangements upon nucleotide exchange [38]. The structural homology of the eIF5B G-domain to EF-Tu and EF-G suggests a related mode of interaction with the ribosome [38], including the mechanism of GTPase activation. The crystal structure of eIF5B has been used to model IF2 in EM reconstructions of bacterial initiation complexes [39,40,41,42] but the conformational changes are larger than anticipated based on the crystal structures. In the 70S:IF1:IF2:IF3:tRNA complex (PDB: 1ZO1), the G-domain is located near the Sarcin–Ricin-Loop, in a position similar to EF-Tu (PDB: 2WRN), while the Cterminal b barrel contacts the aminoacceptor end of initiator tRNA in the ribosomal P-site [39] (Figure 3). This orientation corresponds to the GTP-bound form and agrees well with hydroxyl radical probing data of eIF5B on the 80S ribosome obtained in presence of a nonhydrolysable GTP analog [43]. Compared to the canonical P-site tRNA conformation (PDB: 2WDK), the elbow of the initiator tRNA is rotated toward the small subunit head and the E-site (Figure 3a), and a very similar conformation is also observed in the 30S initiation complexes [41,42]. As established in a different EM study following GTP hydrolysis and Pi release, domain IV resides in the ribosomal A-site and the G-domain is shifted downward [40,41]. Taken together, these results suggest that eIF5B mediates subunit joining by providing additional contacts between the subunits and by positioning the tRNA. During subunit joining its G-domain is optimally positioned to interact with the SRL region of the large subunit and upon GTP hydrolysis the factor undergoes significant conformational changes and ultimately dissociates along with eIF1A [37] (Scheme 1 panel 5).
Initiation factor 6 Initiation factor 5B In contrast to bacteria, translation initiation in eukaryotes requires two GTPases, eIF2 and eIF5B, for delivery of initiator tRNA and subunit joining, respectively, [1,2]. While bacterial IF2 recruits initiator tRNA to the 30S subunit [33], its eukaryotic ortholog eIF5B mainly functions in subunit joining [34] (Scheme 1 panel 5). An interaction with the CTT of eIF1A is required for recruitment and activation of eIF5B [35,36] and GTP hydrolysis by eIF5B enables the release of eIF5B and eIF1A upon subunit joining [37]. The crystal structure of eIF5B has been determined in isolation. The core of eIF5B (PDB: 1G7R, 1G7S, 1G7T) Current Opinion in Structural Biology 2012, 22:1–10
Initiation factor 6 is shared by eukaryotes (eIF6, Tif6p in yeast) and archaea (aIF6). It binds to a conserved site on the large ribosomal subunit in proximity to the Sarcin– Ricin-Loop and sterically precludes joining of the small subunit [10,44,45] (Figures 1b and 3b). The partitioning of eIF6 between nucleus and cytoplasm is controlled by phosphorylation. A nuclear pool of eIF6 is required during ribosome biogenesis and eIF6 remains bound to the large subunit during nuclear export [46]. Release of eIF6 is catalyzed by the GTPase Efl1 and the SBDS protein (sdo1p in yeast) [47] (Scheme 1 panel 8). In analogy to other translational GTPases, Efl1 may be activated through interactions with the ribosome, which www.sciencedirect.com
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Structural insights into eukaryotic ribosomes and the initiation of translation Voigts-Hoffmann, Klinge and Ban 7
Figure 3
(b)
(a)
central protuberance
tRNAiMet
beak
elF5B domain 4 tRNAiMet
P-stalk
C
mRNA
62Å
SRL
elF5B domain 4
elF1A
helical linker elF5B domains 1-3
elF5B domains 1-3
h44 right foot
90º
90º
left foot Current Opinion in Structural Biology
Homology models of eIF5B on the small and large subunit. (a) Models of eIF5B (blue), eIF1A (yellow), mRNA (red) and initiator tRNA (violet) docked on the small ribosomal subunit (PDBs: 3U5B, 3U5C). The binding interface of eIF1A and eIF5 is indicated by spheres and the yellow circle shows the region accessible to the flexible C-terminal tail of eIF1A in the most extended conformation (62 A˚) according to the NMR structure (PDB: 1D7Q). The distance between the truncated C-terminus of eIF1A and the residue with the strongest NMR interaction corresponds to (48 A˚) [36]. Initiation factor eIF1A (PDB: 2OQK) has been positioned on the small subunit according to the bacterial ortholog IF1 (PDB: 1HR0). The N-terminal and C-terminal regions of eIF5B (PDB: 1G7R) were individually positioned on the small subunit according to the EM model of the bacterial ortholog IF2 (PDB: 1ZO1). The position of initiator tRNA is based on the bacterial initiation complex. (PDB: 1ZO1). Ribosomal proteins are represented as light blue surfaces, ribosomal RNA as gray and white spheres. (b) Models of eIF5B and initiator tRNA on the large ribosomal subunit (PDBs: 3U5D, 3U5E) derived as described above. The relative position of the large subunit is based on the 80S ribosome (PDBs: 3U5B–3U5E).
may lead to conformational rearrangement that triggers the dissociation of eIF6 via SBDS [44,47]. Cytoplasmic eIF6 is required for the stimulation of translation in response to growth factor signaling in mammals [48], possibly because it prevents the formation of nontranslating 80S and regulates translation through 60S availability [6]. However, the mechanism by which the anti-association activity of eIF6 is regulated remains to be established.
Discussion The recent crystal structures of the 40S:eIF1 (PDB: 2XZM) and 60S:eIF6 (PDBs: 4A17, 4A19) complexes, eIF2-bound initiator tRNA (PDB: 3V11) and the 80S ribosome (PDBs: 3U5B, 3U5C), represent a significant progress toward a more complete understanding of translation in eukaryotes [7,8,9,10]. Compared to bacterial and archaeal ribosomes, eukaryotic ribosomes contain many additional proteins and rRNA elements, which form a network of interactions. With the availability of atomic structures, the functional roles of these www.sciencedirect.com
additional elements may now be scrutinized, using biochemical and genetic techniques (Figure 1). Combined with the structural data on bacterial initiation complexes and the structures of individual initiation factors or subcomplexes (Tables 1 and 2), initial models of the eukaryotic initiation complexes are beginning to emerge (Figures 2 and 3). Nevertheless, central mechanistic questions remain. For instance, it has not been determined how eIF1 and eIF5 control the GTPase activity and the release of Pi from eIF2, which commits the ribosome to initiation at a particular start codon. Initiation factors eIF1, eIF2b and the N-terminal domain of eIF5 are structurally related, sharing similar a/b folds [23]. This structural similarity may have several implications for the mechanism of start codon selection, including GTPase activation, Pi release and dissociation of eIF2 [5,19,23]. For instance, the displacement of eIF2b from the eIF2g subunit by the structurally related eIF5 NTD may stimulate the hydrolysis of eFI2g-bound GTP. Upon start codon recognition, eIF1 dissociates, which may allow the eIF5 NTD Current Opinion in Structural Biology 2012, 22:1–10
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8 Proteins
Table 2 List of structures. Experimental details and accession codes of structures discussed in the text or used for figure generation as described in the respective figure legend. A complete pymol session including the coordinate files of 40S, 60S and initiation factors as well as a pymol script for the superpositions is available for download on http://www.mol.biol.ethz.ch/groups/ban_group Structure
Source
Method
Res. [A˚]
40S:eIF1 40S:eIF1:eIF1A 60S:eIF6 80S:Stm1 30S:IF1 70S:tRNAs 70S:EF-Tu:tRNA 70S:IF1:IF2:IF3:mRNA:tRNA 30S:IF1:IF2:mRNA:tRNA 30S:IF1:IF2:IF3:mRNA:tRNA eIF1A eIF1A aIF5B aIF2:GDPNP:tRNA aIF2 (a, b, g)
euk euk euk euk bact bact bact bact bact bact euk euk arch arch arch
X-ray Cryo-EM X-ray X-ray X-ray X-ray X-ray EM EM EM X-ray NMR X-ray X-ray X-ray
3.9 22–25 3.5 3.0 3.2 3.5 3.6 13.8 8.7 18.3 1.8 n/a 2.0 5.0 3.2
Accession codes PDB: 2XZM EMD-1346, EMD-1347, EMD-1348, EMD-1349 PDB: 4A17, PDB: 4A19 PDB: 3U5B, PDB: 3U5C, PDB: 3U5D, PDB: 3U5E PDB: 1HR0 PDB: 2WDK, PDB: 2WDL PDB: 2WRN, PDB: 2WRO PDB: 1ZO1, PDB: 1ZO3, EMD-1248, EMD-1249 EMD-1523 EMD-1771 PDB: 2OQK PDB: 1D7Q PDB: 1G7R PDB: 3V11 PDB: 2QMU
n/a: not applicable.
to dissociate from eIF2g and bind to the 40S site previously occupied by eIF1, enabling Pi release from eFI2g. Whereas eIF5 does stimulate the dissociation of eIF1 upon start codon recognition there is no evidence for direct binding of eIF5 to the 40S and, therefore, the mechanistic relevance of the structural similarity between the factors remains to be established [5,19]. Further important aspects of start codon selection include the structural roles of eIF1 and eIF1A including its NTT and CTT. Crystal structures of eIF5B, the GTPase that mediates subunit joining, reveal modest rearrangements depending on the nucleotide binding state, which are amplified by a long helical linker between the structural core and the tRNA binding domain [38]. While the structural integrity of this helical linker indeed affects initiator tRNA binding and subunit joining [49], the crystal structures do not fully account for the domain arrangement in EM reconstructions of ribosomal IF2 complexes corresponding to the state before GTP hydrolysis [38– 42]. Small angle X-ray scattering studies suggest that contacts with initiator tRNA and the ribosome are required for the GTPase to assume its active conformation [50]. The models of eukaryotic initiation complexes described herein are based on the recently determined crystal structures of the eukaryotic ribosome, cryo-EM reconstructions of bacterial initiation complexes and structures of individual initiation factors. They provide first insights into how larger PICs may be organized. While these data may guide the design of biochemical, genetic and spectroscopic experiments, further structures of initiation complexes trapped at different stages of the process will Current Opinion in Structural Biology 2012, 22:1–10
ultimately be required to better understand the molecular mechanisms that govern eukaryotic translation initiation.
Acknowledgements We thank Daniel Bo¨hringer for the superposition of EM structures and Jan Erzberger for his support in drafting the article and critical reading of the manuscript.
Appendix A. Supplementary data Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/ j.sbi.2012.07.010.
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Please cite this article in press as: Voigts-Hoffmann F, et al.: Structural insights into eukaryotic ribosomes and the initiation of translation, Curr Opin Struct Biol (2012), http://dx.doi.org/10.1016/ j.sbi.2012.07.010