Virus propagation relies on the host translational apparatus. To efficiently compete with host mRNAs and engage in translation under stress, some viral mRNAs undergo cap-independent translation. To this end, internal ribosome entry site (IRES) RNAs are employed (reviewed in Deforges et al. (2015), Jackson et al. (2010), Lozano and Martinez-Salas (2015). An IRES is located at the 5’ untranslated region of the viral mRNA, preceding an open reading frame (ORF). To initiate translation, a structured IRES RNA interacts with the 40S subunit or the 80S ribosome, resulting in precise positioning of the downstream start codon in the small 40S subunit. The canonical scenario of cap-dependent and IRES-dependent initiation involves positioning of the AUG start codon and the initiator tRNA^Met in the ribosomal peptidyl-tRNA (P) site, facilitated by interaction with initiation factors (Jackson et al., 2010). Subsequent binding of an elongator aminoacyl-tRNA to the ribosomal A site transitions the initiation complex into the elongation cycle of translation. Upon peptide bond formation, the two tRNAs and their respective mRNA codons translocate from the A and P to P and E (exit) sites, freeing the A site for the next elongator tRNA.

An unusual strategy of initiation is used by intergenic-region (IGR) IRESs found in Dicistroviridae arthropod-infecting viruses. These include shrimp-infecting Taura syndrome virus (TSV; Cevallos and Sarnow, 2005; Hatakeyama et al., 2004), and insect viruses Plautia stali intestine virus (PSIV; Nishiyama et al. (2003), Sasaki and Nakashima (1999)) and Cricket paralysis virus (CrPV; Jan et al. (2001), Wilson et al. (2000)). The IGR IRES mRNAs do not contain an AUG start codon. The IGR-IRES-driven initiation does not involve initiator tRNA^Met and initiation factors (Jan et al., 2001; Sasaki and Nakashima, 1999; Wilson et al., 2000). As such, this group of IRESs represents the most streamlined mechanism of eukaryotic translation initiation. A recent demonstration of bacterial translation initiation by an IGR IRES (Colussi et al., 2015) indicates that the IRESs take advantage of conserved structural and dynamic properties of the ribosome. Early electron cryo-microscopy (cryo-EM) studies have found that the CrPV IRES packs in the ribosome intersubunit space (Schuler et al., 2006; Spahn et al., 2004b). Recent cryo-EM structures of ribosome-bound TSV IRES (Koh et al., 2014) and CrPV IRES (Fernandez et al., 2014) revealed that IGR IRESs position the ORF by mimicking a translating ribosome bound with tRNA and mRNA. The ~200-nt IRES RNAs span from the A site beyond the E site. A conserved tRNA-mRNA–like structural element of pseudoknot I (PKI; Costantino et al., 2008) interacts with the decoding center in the A site of the 40S subunit (Fernandez et al., 2014; Koh et al., 2014). The codon-anticodon-like helix of PKI is stabilized by interactions with the universally conserved decoding-center nucleotides G577, A1755 and A1756 (G530, A1492 and A1493 in E. coli 16S ribosomal RNA, or rRNA). The downstream initiation codon—coding for alanine—is placed in the mRNA tunnel, preceding the decoding center. PKI of IGR IRESs therefore mimics an A-site elongator tRNA interacting with an mRNA sense codon, but not a P-site initiator tRNA^Met and the AUG start codon.

How this non-canonical initiation complex transitions to the elongation step is not fully understood. For a cognate aminoacyl-tRNA to bind the first viral mRNA codon, PKI has to be translocated from the A site, so that the first codon can be presented in the A site. A cryo-EM structure of the ribosome bound with a CrPV IRES and release factor eRF1 occupying the A site provided insight into the post-translocation state (Muhs et al., 2015). In this structure, PKI is positioned in the P site and the first mRNA codon is located in the A site. How the large IRES RNA translocates within the ribosome, allowing PKI translocation from the A to P site is not known.

The structural similarity of PKI and the tRNA anticodon stem loop (ASL) bound to a codon suggests that their mechanisms of translocation are similar to some extent. Translocation of the IRES or tRNA-mRNA requires eukaryotic elongation factor 2 (eEF2) (Jan et al., 2003; Pestova and Hellen, 2003), a structural and functional homolog of the well-studied bacterial EF-G (Czworkowski et al., 1994; Evarsson et al., 1994). Pre-translocation tRNA-bound ribosomes contain a peptidyl- and deacyl-tRNA, both base-paired to mRNA codons in the A and P sites (termed 2tRNA•mRNA complex). Translocation of 2tRNA•mRNA involves two major large-scale ribosome rearrangements (Figure 1—figure supplement 1) (reviewed in Ling and Ermolenko, (2016)). First, studies of bacterial ribosomes showed that a ~10° rotation of the small subunit relative to the large subunit, known as intersubunit rotation, or ratcheting (Frank and Agrawal, 2000), is required for translocation (Horan and Noller, 2007). Intersubunit rotation occurs spontaneously upon peptidyl transfer, and is coupled with formation of hybrid tRNA states (Agirrezabala et al., 2008; Blanchard et al., 2004; Cornish et al., 2008; Ermolenko et al., 2007; Julián et al., 2008; Moazed and Noller, 1989). In the rotated pre-translocation ribosome, the peptidyl-tRNA binds the A site of the small subunit with its ASL and the P site of the large subunit with the CCA 3’ end (A/P hybrid state). Concurrently, the deacyl-tRNA interacts with the P site of the small subunit and the E site of the large subunit (P/E hybrid state). The ribosome can undergo spontaneous, thermally-driven forward-reverse rotation (Cornish et al., 2008) that shifts the two tRNAs between the hybrid and 'classical' states while the anticodon stem loops remain non-translocated. Binding of EF-G next to the A site and reverse rotation of the small subunit results in translocation of both ASLs on the small subunit (Ermolenko and Noller, 2011). EF-G is thought to 'unlock' the pre-translocation ribosome (Savelsbergh et al., 2003; Spirin, 1969), allowing movement of the 2tRNA•mRNA complex, however the structural details of this unlocking are not known.

The second large-scale rearrangement involves rotation, or swiveling, of the head of the small subunit relative to the body. The head can rotate by up to ~20° around the axis nearly orthogonal to that of intersubunit rotation, in the absence of tRNA (Schuwirth et al., 2005) or in the presence of a single P/E tRNA and eEF2 (Taylor et al., 2007) or EF-G (Ratje et al., 2010). Förster resonance energy transfer (FRET) data suggest that head swivel of the rotated small subunit facilitates EF-G-mediated movement of 2tRNA•mRNA (Guo and Noller, 2012). Structures of the 70S•EF-G complex bound with two nearly translocated tRNAs (Ramrath et al., 2013; Zhou et al., 2014), exhibit a large 18° to 21° head swivel in a mid-rotated subunit, whereas no head swivel is observed in the fully rotated pre-translocation or in the non-rotated post-translocation 70S•2tRNA•EF-G structures (Brilot et al., 2013; Gao et al., 2009). The structural role of head swivel is not fully understood. The head swivel was proposed to facilitate transition of the tRNA from the P to E site by widening a constriction between these sites on the 30S subunit (Schuwirth et al., 2005). This widening allows the ASL to sample positions between the P and E sites (Ratje et al., 2010). Whether and how the head swivel mediates tRNA transition from the A to P site remains unknown.

We sought to address the following questions by structural visualization of 80S•IRES•eEF2 translocation complexes: (1) How does a large IRES RNA move through the restricted intersubunit space, bringing PKI from the A to P site of the small subunit? (2) How does eEF2 mediate IRES translocation? (3) Does IRES translocation involve large rearrangements in the ribosome, similar to tRNA translocation? (4) What, if any, is the mechanistic role of 40S head rotation in IRES translocation? We used cryo-EM to visualize 80S•TSV IRES complexes formed in the presence of eEF2•GTP and the translation inhibitor sordarin, which stabilizes eEF2 on the ribosome. Although the mechanism of sordarin action is not fully understood, the inhibitor does not affect the conformation of eEF2•GDPNP on the ribosome (Taylor et al., 2007), rendering it an excellent tool in translocation studies. Maximum-likelihood classification using FREALIGN (Lyumkis et al., 2013) identified five IRES-eEF2-bound ribosome structures within a single sample (Figures 1 and 2). The structures differ in the positions and conformations of ribosomal subunits (Figures 1b and 2), IRES RNA (Figures 3 and 4) and eEF2 (Figures 5 and 6). This ensemble of structures allowed us to reconstruct a sequence of steps in IRES translocation induced by eEF2.

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Figure 1—source data 1

Structure refinement statistics for Structures I, II, III, IV, V.

https://doi.org/10.7554/eLife.14874.003

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We used single-particle cryo-EM and maximum-likelihood image classification in FREALIGN to obtain three-dimensional density maps from a single specimen. The translocation complex was formed using S. cerevisiae 80S ribosomes, Taura syndrome virus IRES, and S. cerevisiae eEF2 in the presence of GTP and the eEF2-binding translation inhibitor sordarin. Unsupervised cryo-EM data classification was combined with the use of three-dimensional and two-dimensional masking around the ribosomal A site (Figure 1—figure supplement 2). This approach revealed five 80S•IRES•eEF2•GDP structures at average resolutions of 3.5 to 4.2 Å, sufficient to locate IRES domains and to resolve individual residues in the core regions of the ribosome and eEF2 (Figures 3c,d, and 5f,h; see also Figure 1—figure supplement 2 and Figure 5—figure supplement 2), including the post-translational modification diphthamide 699 (Figure 3c).

Our structures represent hitherto uncharacterized translocation complexes of the TSV IRES captured within globally distinct 80S conformations (Figures 1b and 2). We numbered the structures from I to V, according to the position of the tRNA-mRNA-like PKI on the 40S subunit (Figure 2—source data 1). Specifically, PKI is partially withdrawn from the A site in Structure I, and fully translocated to the P site in Structure V (Figure 4; see also Figure 3—figure supplement 1). Thus Structures I to IV represent different positions of PKI between the A and P sites (Figure 2—source data 1), suggesting that these structures describe intermediate states of translocation. Structure V corresponds to the post-translocation state.

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Figure 2—source data 1

Measurements for conformations and positions in Structures I through V.

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Changes in ribosome conformation and eEF2 positions are coupled with IRES movement through the ribosome

Intersubunit rotation

Using the post-translocation S. cerevisiae 80S ribosome bound with the P and E site tRNAs as a reference (80S•2tRNA•mRNA), in which both the subunit rotation and the head-body swivel are 0°(Svidritskiy et al., 2014), we found that the ribosome adopts four globally distinct conformations in Structures I through V (Figure 1b; see also Figure 1—figure supplement 1 and Figure 2—source data 1). Structure I comprises the most rotated ribosome conformation (~10°), characteristic of pre-translocation hybrid-tRNA states. From Structure I to V, the body of the small subunit undergoes backward (reverse) rotation (Figure 2b; see also Figure 1—figure supplement 2 and Figure 2—figure supplement 1). Structures II and III are in mid-rotation conformations (~5°). Structure IV adopts a slightly rotated conformation (~1°). Structure V is in a nearly non-rotated conformation (0.5°), very similar to that of post-translocation ribosome-tRNA complexes (Gao et al., 2009; Korostelev et al., 2006; Selmer et al., 2006; Svidritskiy et al., 2014). Thus, intersubunit rotation of ~9° from Structure I to V covers a nearly complete range of relative subunit positions, similar to what was reported for tRNA-bound yeast (Svidritskiy et al., 2014; Taylor et al., 2007), bacterial (Agirrezabala et al., 2008; Fischer et al., 2010; Julián et al., 2008) and mammalian (Budkevich et al., 2011) ribosomes.

40S head swivel

The pattern of 40S head swivel between the structures is different from that of intersubunit rotation (Figures 2c and d; see also Figure 2—source data 1). As with the intersubunit rotation, the small head swivel (~1°) in the non-rotated Structure V is closest to that in the 80S•2tRNA•mRNA post-translocation ribosome (Svidritskiy et al., 2014). However in the pre-translocation intermediates (from Structure I to IV), the beak of the head domain first turns toward the large subunit and then backs off (Figure 2—figure supplement 1). This movement reflects the forward and reverse swivel. The head samples a mid-swiveled position in Structure I (12°), then a highly-swiveled position in Structures II and III (17°) and a less swiveled position in Structure IV (14°). The maximum head swivel is observed in the mid-rotated complexes II and III, in which PKI transitions from the A to P site, while eEF2 occupies the A site partially. By comparison, the similarly mid-rotated (4°) 80S•TSV IRES initiation complex, in the absence of eEF2 (Koh et al., 2014), adopts a mid-swiveled position (~10°) (Figure 2c). These observations suggest that eEF2 is necessary for inducing or stabilizing the large head swivel of the 40S subunit characteristic for IRES translocation intermediates.

IRES rearrangements

In each structure, the TSV IRES adopts a distinct conformation in the intersubunit space of the ribosome (Figures 3 and 4). The IRES (nt 6758–6952) consists of two globular parts (Figure 3a): the 5’-region (domains I and II, nt 6758–6888) and the PKI domain (domain III, nt 6889–6952). We collectively term domains I and II the 5’ domain. The PKI domain comprises PKI and stem loop 3 (SL3), which stacks on top of the stem of PKI (Koh et al., 2014). The ⁶⁹⁵³GCU triplet immediately following the PKI domain is the first codon of the open reading frame. In the eEF2-free 80S•IRES initiation complex (INIT) (Koh et al., 2014), the bulk of the 5’-domain (nt. 6758–6888) binds near the E site, contacting the ribosome mostly by means of three protruding structural elements: the L1.1 region and stem loops 4 and 5 (SL4 and SL5). In Structures I to IV, these contacts remain as in the initiation complex (Figure 1a). Specifically, the L1.1 region interacts with the L1 stalk of the large subunit, while SL4 and SL5 bind at the side of the 40S head and interact with proteins uS7, uS11 and eS25 (Figure 3—figure supplement 2 and Figure 3—figure supplement 3; ribosomal proteins are termed according to Ban et al., 2014). In Structures I-IV, the minor groove of SL4 (at nt 6840–6846) binds next to an α-helix of uS7, which is rich in positively charged residues (K212, K213, R219 and K222). The tip of SL4 binds in the vicinity of R157 in the β-hairpin of uS7 and of Y58 in uS11. The minor groove of SL5 (at nt 6862–6868) contacts the positively charged region of eS25 (R49, R58 and R68) (Figure 3—figure supplement 4). In Structure V, however, the density for SL5 is missing suggesting that SL5 is mobile, while weak SL4 density suggests that SL4 is shifted along the surface of uS7, ~20 Å away from its initial position (Figure 3—figure supplement 2c). The L1.1 region remains in contact with the L1 stalk (Figure 3—figure supplement 3).

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The shape of the IRES changes considerably from the initiation state to Structures I through V, from an extended to compact to extended conformation (Figure 4; see also Figure 3—figure supplement 2a). Because in Structures I to IV the PKI domain shifts toward the P site, while the 5’ remains unchanged near the E site, the distance between the domains shortens (Figure 4). In the 80S•IRES initiation state (Koh et al., 2014), the A-site-bound PKI is separated from SL4 by almost 50 Å (Figure 4). In Structures I and II, the PKI is partially retracted from the A site and the distance from SL4 shortens to ~35 Å. As PKI moves toward the P site in Structures III and IV, the PKI domain approaches to within ~25 Å of SL4. Because the 5’-domain in the following structure (V) moves by ~20 Å along the 40S head, the IRES returns to an extended conformation (~45 Å) that is similar to that in the 80S•IRES initiation complex.

Figure 4

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Rearrangements of the IRES involve restructuring of several interactions with the ribosome. In Structure I, SL3 of the PKI domain is positioned between the A-site finger (nt 1008–1043 of 25S rRNA) and the P site of the 60S subunit, comprising helix 84 of 25S rRNA (nt. 2668–2687) and protein uL5 (Figure 3—figure supplement 6). This position of SL3 is ~25 Å away from that in the 80S•IRES initiation state (Koh et al., 2014), in which PKI and SL3 closely mimic the ASL and elbow of the A-site tRNA, respectively (Koh et al., 2014). As such, the transition from the initiation state to Structure I involves repositioning of SL3 around the A-site finger, resembling the transition between the pre-translocation A/P and A/P* tRNA (Brilot et al., 2013). The second set of major structural changes involves interaction of the P site region of the large subunit with the hinge point of the IRES bending between the 5´ domain and the PKI domain (nt. 6886–6890). In the highly bent Structures III and IV, the hinge region interacts with the universally conserved uL5 and the C-terminal tail of eL42 (Figure 3—figure supplement 7). However, in the extended conformations, these parts of the IRES and the 60S subunit are separated by more than 10 Å, suggesting that an interaction between them stabilizes the bent conformations but not the extended ones. Another local rearrangement concerns loop 3, also known as the variable loop region (Ruehle et al., 2015; Ren et al., 2014; Au and Jan, 2012), which connects the ASL- and mRNA-like parts of PKI. This loop is poorly resolved in Structures I through IV, suggesting conformational flexibility in agreement with structural studies of the isolated PKI (Costantino et al., 2008; Zhu et al., 2011) and biochemical studies of unbound IRESs (Jan and Sarnow, 2002; Pfingsten et al., 2010). In Structure V, loop 3 is bound in the 40S E site and the backbone of loop 3 near the codon-like part of PKI (at nt. 6945–6946) interacts with R148 and R157 in β-hairpin of uS7. The interaction of loop 3 backbone with uS7 resembles that of the anticodon-stem loop of E-site tRNA in the post-translocation 80S•2tRNA•mRNA structure (Figure 3—figure supplement 5) (Svidritskiy et al., 2014). Ordering of loop 3 suggests that this flexible region contributes to the stabilization of the PKI domain in the post-translocation state. This interpretation is consistent with the recent observation that alterations in loop 3 of the CrPV IRES result in decreased efficiency of translocation (Ruehle et al., 2015).

eEF2 structures

Elongation factor eEF2 in all five structures is bound with GDP and sordarin (Figure 5). The elongation factor consists of three dynamic superdomains (Jorgensen et al., 2003): an N-terminal globular superdomain formed by the G (GTPase) domain (domain I) and domain II; a linker domain III; and a C-terminal superdomain comprising domains IV and V (Figure 5a). Domain IV extends from the main body and is critical for translocation catalyzed by eEF2 or EF-G. ADP-ribosylation of eEF2 at the tip of domain IV (Davydova and Ovchinnikov, 1990; Nygard and Nilsson, 1990) or deletion of domain IV from EF-G (Martemyanov and Gudkov, 1999; Rodnina et al., 1997) abrogate translocation. In post-translocation-like 80S•tRNA•eEF2 complexes, domain IV binds in the 40S A site, suggesting direct involvement of domain IV in translocation of tRNA from the A to P site (Spahn et al., 2004a; Taylor et al., 2007). GDP in our structures is bound in the GTPase center (Figures 5d, e and f) and sordarin is sandwiched between the β-platforms of domains III and V (Figures 5g and h), as in the structure of free eEF2•sordarin complex (Jorgensen et al., 2003).

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The global conformations of eEF2 (Figure 5a) are similar in these structures (all-atom RMSD ≤ 2 Å), but the positions of eEF2 relative to the 40S subunit differ substantially as a result of 40S subunit rotation (Figure 2—source data 1). From Structure I to V, eEF2 is rigidly attached to the GTPase-associated center of the 60S subunit. The GTPase-associated center comprises the P stalk (L11 and L7/L12 stalk in bacteria) and the sarcin-ricin loop (SRL, nt 3012–3042). The tips of 25S rRNA helices 43 and 44 of the P stalk (nucleotides G1242 and A1270, respectively) stack on V754 and Y744 of domain V. An αββ motif of the eukaryote-specific protein P0 (aa 126–154) packs in the crevice between the long α-helix D (aa 172–188) of the GTPase domain and the β-sheet region (aa 246–263) of the GTPase domain insert (or G’ insert) of eEF2 (secondary-structure nomenclatures for eEF2 and EF-G (Czworkowski et al., 1994) are the same). Although the P/L11 stalk is known to be dynamic (Korostelev et al., 2008; Taylor et al., 2012), its position remains unchanged from Structure I to V: all-atom root-mean-square differences for the 25S rRNA of the P stalk (nt 1223–1286) are within 2.5 Å. However, with respect to its position in the 80S•IRES complex in the absence of eEF2 and in the 80S•2tRNA•mRNA complex, the P stalk is shifted by ~13 Å toward the A site (Figure 2d). The sarcin-ricin loop interacts with the GTP-binding site of eEF2 (Figures 5d and f). While the overall mode of this interaction is similar to that seen in 70S•EF-G crystal structures (Chen et al., 2013b; Gao et al., 2009; Pulk and Cate, 2013; Tourigny et al., 2013; Zhou et al., 2013; 2014), there is an important local difference between Structure I and Structures II-V in switch loop I, as discussed below.

There are two modest but noticeable domain rearrangements between Structures I and V. Unlike in free eEF2, which can sample large movements of at least 50 Å of the C-terminal superdomain relative to the N-terminal superdomain (Figure 5c) (Jorgensen et al., 2003), eEF2 undergoes moderate repositioning of domain IV (~3 Å; Figure 5a) and domain III (~5 Å; Figure 6d). This limited flexibility of the ribosome-bound eEF2 is likely the result of simultaneous fixation of eEF2 superdomains, via domains I and V, by the GTPase-associated center of the large subunit. Domain IV of eEF2 binds at the 40S A site in Structures I to V but the mode of interaction differs in each complex (Figure 6). Because eEF2 is rigidly attached to the 60S subunit and does not undergo large inter-subunit rearrangements, gradual entry of domain IV into the A site between Structures I and V is due to 40S subunit rotation and head swivel. eEF2 settles into the A site from Structure I to V, as the tip of domain IV shifts by ~10 Å relative to the body and by ~20 Å relative to the swiveling head. Modest intra-eEF2 shifts of domain IV between Structures I to V outline a stochastic trajectory (Figure 5a), consistent with local adjustments of the domain in the A site. At the central region of eEF2, domains II and III contact the 40S body (mainly at nucleotides 48–52 and 429–432 of 18S rRNA helix 5 and uS12). From Structure I to V, these central domains migrate by ~10 Å along the 40S surface (Figure 6c). Comparison of eEF2 conformations reveals that in Structure V, domain III is displaced as a result of interaction with uS12, as discussed below.

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In summary, between Structures I and V, a step-wise translocation of PKI by ~15 Å from the A to P site - within the 40S subunit – occurs simultaneously with the ~11 Å side-way entry of domain IV into the A site coupled with ~3 to 5 Å inter-domain rearrangements in eEF2. These shifts occur during the reverse rotation of the 40S body coupled with the forward-then-reverse head swivel. To elucidate the detailed structural mechanism of IRES translocation and the roles of eEF2 and ribosome rearrangements, we describe in the following sections the interactions of PKI and eEF2 with the ribosomal A and P sites in Structures I through V (Figure 2g; see also Figure 1—figure supplement 1).

Structure I represents a pre-translocation IRES and initial entry of eEF2 in a GTP-like state

In the fully rotated Structure I, PKI is shifted toward the P site by ~3 Å relative to its position in the initiation complex but maintains interactions with the partially swiveled head. At the head, C1274 of the 18S rRNA (C1054 in E. coli) base pairs with the first nucleotide of the ORF immediately downstream of PKI. The C1274:G6953 base pair provides a stacking platform for the codon-anticodon–like helix of PKI. We therefore define C1274 as the foundation of the 'head A site'. Accordingly, we use U1191 (G966 in E. coli) and C1637 (C1400 in E. coli) as the reference points of the 'head P site' and 'body P site' (Figure 2g), respectively, because these nucleotides form a stacking foundation for the fully translocated mRNA-tRNA helix in tRNA-bound structures (Korostelev et al., 2006; Selmer et al., 2006; Svidritskiy et al., 2014) and in our post-translocation Structure V discussed below.

The interaction of PKI with the 40S body is substantially rearranged relative to that in the initiation state. In the latter, PKI is stabilized by interactions with the universally conserved decoding-center nucleotides G577, A1755 and A1756 ('body A site'), as in the A-site tRNA bound complexes (Koh et al., 2014). In Structure I, PKI does not contact these nucleotides (Figures 2g and 7).

Figure 7

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The position of eEF2 on the 40S subunit of Structure I is markedly distinct from those in Structures II to V. The translocase interacts with the 40S body but does not contact the head (Figures 5b and 6a; Figure 5—figure supplement 1). Domain IV is partially engaged with the body A site. The tip of domain IV is wedged between PKI and decoding-center nucleotides A1755 and A1756, which are bulged out of h44. This tip contains the histidine-diphthamide triad (H583, H694 and Diph699), which interacts with the codon-anticodon-like helix of PKI and A1756 (Figure 7). Histidines 583 and 694 interact with the phosphate backbone of the anticodon-like strand (at G6907 and C6908). Diphthamide is a unique posttranslational modification conserved in archaeal and eukaryotic EF2 (at residue 699 in S. cerevisiae) and involves addition of a ~7-Å long 3-carboxyamido-3-(trimethylamino)-propyl moiety to the histidine imidazole ring at CE1. The trimethylamino end of Diph699 packs over A1756 (Figure 7). The opposite surface of the tail is oriented toward the minor-groove side of the second base pair of the codon-anticodon helix (G6906:C6951). Thus, in comparison with the initiation state, the histidine-diphthamide tip of eEF2 replaces the codon-anticodon–like helix of PKI. The splitting of the interaction of A1755-A1756 and PKI is achieved by providing the histidine-diphthamine tip as a binding partner for both A1756 and the minor groove of the codon-anticodon helix (Figure 7).

Unlike in Structures II to V, the conformation of the eEF2 GTPase center in Structure I resembles that of a GTP-bound translocase (Figure 5e). In translational GTPases, switch loops I and II are involved in the GTPase activity (reviewed in Voorhees and Ramakrishnan, (2013)). Switch loop II (aa 105–110), which carries the catalytic H108 (H92 in E. coli EF-G; (Cunha et al., 2013; Holtkamp et al., 2014; Koripella et al., 2015; Salsi et al., 2014) is well resolved in all five structures. The histidine resides next to the backbone of G3028 of the sarcin-ricin loop and near the diphosphate of GDP (Figure 5e). By contrast, switch loop I (aa 50–70 in S. cerevisiae eEF2) is resolved only in Structure I (Figure 5—figure supplement 2). The N-terminal part of the loop (aa 50–60) is sandwiched between the tip of helix 14 (⁴¹⁵CAAA⁴¹⁸) of the 18S rRNA of the 40S subunit and helix A (aa 32–42) of eEF2 (Figure 5d). Bulged A416 interacts with the switch loop in the vicinity of D53. Next to GDP, the C-terminal part of the switch loop (aa 61–67) adopts a helical fold. As such, the conformations of SWI and the GTPase center in general are similar to those observed in ribosome-bound EF-Tu (Voorhees et al., 2010) and EF-G (Pulk and Cate, 2013; Tourigny et al., 2013; Zhou et al., 2013) in the presence of GTP analogs.

1. 1. Priyanka D Abeyrathne
   2. Cha San Koh
   3. Timothy Grant
   4. Nikolaus Grigorieff
   5. Andrei A Korostelev

   (2016) Saccharomyces cerevisiae 80S ribosome bound with elongation factor eEF2-GDP-sordarin and Taura Syndrome Virus IRES, Structure I (fully rotated 40S subunit)

   Publicly available at the RCSB Protein Data Bank (accession no. 5JUO).

   http://www.rcsb.org/pdb/explore/explore.do?structureId=5JUO
2. 1. Priyanka D Abeyrathne
   2. Cha San Koh
   3. Timothy Grant
   4. Nikolaus Grigorieff
   5. Andrei A Korostelev

   (2016) Saccharomyces cerevisiae 80S ribosome bound to elongation factor eEF2-GDP-sordarin and Taura Syndrome Virus IRES, Structure I

   Publicly available at the Electron Microscopy Data Bank (accession no. EMD-6643).

   https://www.ebi.ac.uk/pdbe/entry/emdb/EMD-6643
3. 1. Priyanka D Abeyrathne
   2. Cha San Koh
   3. Timothy Grant
   4. Nikolaus Grigorieff
   5. Andrei A Korostelev

   (2016) Saccharomyces cerevisiae 80S ribosome bound to TSV IRES and eEF2 translation initiation complex, Structure I, Map 2

   Publicly available at the Electron Microscopy Data Bank (accession no. EMD-6648).

   https://www.ebi.ac.uk/pdbe/entry/emdb/EMD-6648
4. 1. Priyanka D Abeyrathne
   2. Cha San Koh
   3. Timothy Grant
   4. Nikolaus Grigorieff
   5. Andrei A Korostelev

   (2016) Saccharomyces cerevisiae 80S ribosome bound to elongation factor eEF2-GDP-sordarin and Taura Syndrome Virus IRES, Structure II (mid-rotated 40S subunit)

   Publicly available at the RCSB Protein Data Bank (accession no. 5JUP).

   http://www.rcsb.org/pdb/explore/explore.do?structureId=5JUP
5. 1. Priyanka D Abeyrathne
   2. Cha San Koh
   3. Timothy Grant
   4. Nikolaus Grigorieff
   5. Andrei A Korostelev

   (2016) Saccharomyces cerevisiae 80S ribosome bound to elongation factor eEF2-GDP-sordarin and Taura Syndrome Virus IRES, Structure II (mid-rotated 40S subunit)

   Publicly available at the Electron Microscopy Data Bank (accession no. EMD-6644).

   https://www.ebi.ac.uk/pdbe/entry/emdb/EMD-6644
6. 1. Priyanka D Abeyrathne
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Author details

1. Priyanka D Abeyrathne

   Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, United States

   Contribution

   PDA, Collected and analyzed cryo-EM data, Drafting or revising the article

   Contributed equally with

   Cha San Koh and Timothy Grant

   Competing interests

   No competing interests declared.

2. Cha San Koh

   RNA Therapeutics Institute, Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, United States

   Contribution

   CSK, Prepared the ribosome•IRES•eEF2 complex, Built and refined structural models, Analysis and interpretation of data, Drafting or revising the article

   Contributed equally with

   Priyanka D Abeyrathne and Timothy Grant

   Competing interests

   No competing interests declared.

   "This ORCID iD identifies the author of this article:" 0000-0002-1579-0362

3. Timothy Grant

   Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, United States

   Contribution

   TG, Assisted with cryo-EM data processing and analyses, Drafting or revising the article

   Contributed equally with

   Priyanka D Abeyrathne and Cha San Koh

   Competing interests

   No competing interests declared.

4. Nikolaus Grigorieff

   Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, United States

   Contribution

   NG, Designed the project, Assisted with cryo-EM data processing and analyses, Drafting or revising the article

   For correspondence

   niko@grigorieff.org

   Competing interests

   NG: Reviewing editor, eLife

5. Andrei A Korostelev

   RNA Therapeutics Institute, Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, United States

   Contribution

   AAK, Designed the project, Built and refined structural models, Analysis and interpretation of data, Drafting or revising the article

   For correspondence

   andrei.korostelev@umassmed.edu

   Competing interests

   No competing interests declared.

   "This ORCID iD identifies the author of this article:" 0000-0003-1588-717X

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