Structural Basis for Ribosome Recycling by RRF and tRNA

Abstract

The bacterial ribosome is recycled into subunits by two conserved proteins, elongation factor G (EF-G) and the ribosome recycling factor (RRF). The molecular basis for ribosome recycling by RRF and EF-G remains unclear. Here, we report the crystal structure of a post-termination Thermus thermophilus 70S ribosome complexed with EF-G, RRF, and two tRNAs at a resolution of 3.5 Å. The deacylated tRNA in the P site moves into a previously unsuspected state of binding (peptidyl/recycling; p/R) that is analogous to that seen during initiation. The terminal end of the p/R-tRNA forms non-favorable contacts with the 50S subunit while RRF wedges next to central inter-subunit bridges, illuminating the active roles of tRNA and RRF in dissociation of ribosomal subunits. The structure uncovers a missing snapshot of tRNA as it transits between the P and E sites, providing insights into the mechanisms of ribosome recycling and tRNA translocation.

Termination of translation occurs when a stop codon on the messenger RNA (mRNA) arrives in the aminoacyl (A) site of the ribosome and is recognized by a release factor, promoting release of the polypeptide chain. The ribosome is left with the mRNA and deacylated tRNAs in the peptidyl (P) and exit (E) sites. In bacteria, post-termination 70S ribosome complexes are recycled by the concerted action of the ribosome recycling factor (RRF) and elongation factor G (EF-G) ¹. Recycling of the ribosome for a new round of translation initiation is an essential step of protein synthesis which entails splitting of the 70S ribosome into the individual 30S and 50S subunits, a process requiring energy from the guanosine triphosphate (GTP) nucleotide ². Despites decades of biochemical studies ^3–9 and more recently the structure determination of ribosome complexes bound to RRF and EF-G ^10–15 that yielded different models for ribosome recycling, the mechanism by which the 70S ribosome is disassembled into subunits remains elusive.

Ribosome recycling proceeds with a splitting rate that is more than fifteen-fold faster in the presence of deacylated tRNA in the P site, suggesting that the tRNA is required for efficient recycling of the ribosome ¹⁶. Models of RRF bound to the ribosome have, however, failed to explain the role of tRNA during ribosome splitting. This is because in all of the available structures of ribosome recycling complexes, there is either no tRNA ¹³, one tRNA bound in the E site ¹⁴, or one bound in the p/E hybrid state which is accompanied by swiveling of the 30S head domain ^11,15,17. In the p/E hybrid state, the deacylated tRNA effectively avoids a steric collision between the acceptor stem and the triple helix bundle domain I of RRF ^11,15,17. However, because the p/E intermediate state of binding also occurs during tRNA translocation ^18–24, and the tRNA eventually exits the ribosome from the E site, its role in ribosome splitting is difficult to reconcile. In agreement with this, dissociation of the ribosome does not involve translocation of mRNA and tRNAs on the ribosome ^4,8.

Recent efforts to stabilize the ribosome in intermediate states of ratcheting and bound to EF-G and tRNAs during translocation for high-resolution structure determination have been successful, providing important insights into the mechanism of protein synthesis ^{18–20,25–27}. These studies revealed that EF-G can adopt a compact conformation to engage the pre-translocation ribosome ^25,28. Subsequent transition in EF-G from the compact to the extended conformation followed by GTP hydrolysis leads to ribosome ratcheting, 30S head domain swiveling, and translocation of mRNA and tRNAs ²⁸. In the presence of GDP or the antibiotic dityromycin, EF-G fails to undergo such conformational change and does not promote tRNA translocation ^25,29,30. In contrast, trapping the ribosome bound to RRF and EF-G before subunit splitting for structure determination remains a challenge.

To investigate the mechanism of ribosome recycling by RRF and EF-G, we reasoned that during ribosome recycling, EF-G may also transiently take the compact conformation, which can be captured in the presence of GDP. Here, we present the crystal structure of a Thermus thermophilus 70S ribosome complex programmed in the post-peptide release state with a stop codon in the A site and bound to RRF, EF-G, and deacylated tRNAs in the presence of GDP. The structure, determined at a resolution of 3.5 Å, represents a ribosome recycling intermediate with two deacylated tRNAs in both the P and E sites (Online Methods and Table 1). This ribosome complex displays a new hybrid state conformation of the P-site tRNA as it is being handed over to the E site by ribosomal protein uL5. The non-favorable contacts between the CCA-end of the P-site tRNA and the 50S subunit of the ribosome, together with the trajectory taken by tRNA as it moves from the P to the E site, provide insights into the mechanisms of ribosome recycling and tRNA translocation.

Table 1.

Data collection and refinement statistics

	70S recycling complex (PDB 6UCQ)
Data collection
Space group	P212121
Cell dimensions
a, b, c (Å)	209.7, 448.7, 624.0
α, β, γ (°)	90, 90, 90
Resolution (Å)	50 – 3.5 (3.71 – 3.50)a
Rsym (%)	33.0 (277.9)
I/σ(I)	4.48 (0.64)
CC 1/2	99.6 (15.2)
Completeness (%)	98.7 (97.3)
Redundancy	4.2 (4.2)
Refinement
Resolution (Å)	50 – 3.5
No. reflections	721,113
Rwork / Rfree	24.4/29.4
No. atoms
Protein	103,654
RNA	197,035
ion	2,902
B factors
Protein	143.7
RNA	131.7
ion	107.1
r.m.s. deviations
Bond lengths (Å)	0.005
Bond angles (°)	0.819

Data set is for a single crystal.

^aValues in parentheses are for highest-resolution shell.

Results

Structure of the ribosome recycling complex.

The ribosome recycling complex was reconstituted by using a fusion between the N-terminal domain of ribosomal protein bL9 and the N-terminus of the T. thermophilus EF-G. The protein fusion, which links Asn74 in protein bL9 to Asp8 in EF-G, previously allowed to obtain high-resolution structures of pre- and post-translocation ribosomes ²⁵. This strategy traps EF-G on the ribosome in the crystal. The addition of the L9-EF-G protein fusion to ribosome complexes promotes crystallization of ribosomes lacking the endogenous bL9 under the same conditions and space group as the wild-type ribosome ²⁷. Similarly, a fusion between protein bL9 and elongation factor 4 (EF-4) revealed new ribosome and tRNA conformational dynamics ^31,32, showing this crystallization strategy to be an excellent tool to capture translational GTPases on the ribosome in different functional states for structural studies.

The structure of the ribosome recycling complex was determined by molecular replacement using as a search model a high-resolution structure of the 70S ribosome with its ligands removed ³³. All components including EF-G, RRF, mRNA, and two tRNAs were immediately visible in the F_obs – F_calc difference Fourier map, suggesting that we have captured a complete ribosome recycling complex (Fig. 1a, Extended Data Fig.1a and Supplementary Video 1). The overall conformation of RRF is similar to that seen in previously determined structures ^13–15, with the tip of the triple helix bundle domain I in RRF forming interactions with nucleotide G2253 in 23S rRNA, part of the P-loop (Extended Data Fig. 2a). As expected, EF-G is in a compact conformation with domains III and V packed against domain II of RRF through shape complementarity (Figs 1a and 2a). The ribosome is in an intermediate state of ratcheting with the 30S subunit rotating counter-clockwise by ~2°, while the head domain of the 30S is not swiveled (Extended Data Fig. 1b). The ribosome bound to RRF has been reported to sample both ratcheted and non-ratcheted states ^{5,13–15,17,34–36}. The relatively small amplitude of ratcheting in our ribosome recycling complex agrees with previous cryo-EM reconstruction ¹⁷ and crystal structures ^13,14 that also reported binding of RRF to a non-ratcheted 70S ribosome.

Fig. 1 |. Structure of the 70S ribosome recycling complex.

a, Overall view of the ribosome recycling complex shows the structure of the T. thermophilus 70S ribosome in complex with tRNA^Phe in the peptidyl (P; magenta) and exit (E; orange) sites, the mRNA (cyan), RRF (dark blue) and EF-G (light blue). The 30S subunit is shown in light yellow, and the 50S subunit in light blue. b, Same as in panel a with the ribosome omitted for clarity. Inset: Close-up view of the interaction between the triple helix bundle domain I of RRF and the CCA-end of the deacylated tRNA^Phe through shape complementarity. c, d, Comparison of tRNAs bound in the classic p/P and e/E states (grey) with the tRNAs bound in the P (magenta) and E (orange) sites in this recycling complex. Domain I of RRF is not compatible with a tRNA in the p/P conformation. The p/R-tRNA (magenta) bends by ~30° along the ASL to avoid a steric collision with domain I of RRF, resulting in displacement of the acceptor/T domain by ~22 Å toward the E site.

Fig. 2 |. Interactions between EF-G, RRF and the ribosome.

a, Domains III and V of EF-G form a surface that is complementary to RRF. b, Superposition of ribosome complexes bound to RRF from E. coli (yellow, PDB 4V54 ¹³) and from T. thermophilus (green, PDB 4V5A ¹⁴) with the current recycling complex using the 23S rRNA shows that domain II of RRF (dark blue) inserts deeper into the niche formed between helix H69 and protein uS12. c, Domain II of RRF is wedged in the niche created between the 23S rRNA helix H69 and ribosomal protein uS12. d, Displacement of the 23S rRNA helix H69 created by insertion of domain II of RRF in the niche between H69 and protein uS12 (grey, PDB 4V5A ¹⁴).

Interactions between RRF and EF-G on the ribosome.

The five domains in EF-G form two superdomains, composed of domains I-II and domains III-V, linked together through a flexible hinge region ²⁵. The conformational flexibility in EF-G allows it to take the compact conformation in the non-ratcheted ribosome recycling complex. Domains III and V of EF-G are arranged with respect to each other with an obtuse angle of ~108°, creating a cleft in which domain II of RRF is located (Fig. 2a). This observation agrees with a previous cryo-EM reconstruction in which domains III and V in EF-G bound to a 50S subunit-RRF complex ¹² and to a 70S ribosome ¹¹ formed interactions with domain II of RRF. The structures also explain data from chimeric constructs of RRF and EF-G that delineated domain II of RRF as an essential element for interaction with EF-G and for ribosome splitting ³⁷. In contrast with the compact EF-G in our structure in which domain IV is directed away from RRF, domain IV of EF-G lies on top of RRF in the cryo-EM reconstructions ^11,12,35. In these studies, the ribosome is in the fully ratcheted state while in our complex, the ribosome is only partially ratcheted. This may explain the difference in the location of domain IV of EF-G and suggests we have trapped a different state.

A comparison of our structure with that of a RRF-ribosome complex in the absence of EF-G ¹³ shows that while the triple helix bundle domain I of RRF occupies the same location, binding of EF-G causes domain II of RRF to rotate by ~28° toward the 23S rRNA helix H69 wedging between ribosomal protein uS12 and H69 (Fig. 2b,c and Extended Data Fig. 2b). The accompanied movement of the tip of H69 by ~8 Å toward the 16S rRNA helix h44 creates a niche into which domain II of RRF is located (Fig. 2d). Similar rotational movement of domain II of RRF toward the central bridge between 30S and 50S subunits, B2a, was visualized by time-resolved cryo-EM reconstructions of post-termination 70S ribosome complexes bound to RRF and EF-G ¹¹.

The P-site tRNA is in a distinct state of binding.

The most remarkable feature in this ribosome complex is the binding state of the deacylated tRNA in the P site. To avoid a steric collision with RRF, the P-site tRNA^Phe bends by ~30° along the anticodon stem loop (ASL) helical axis, such that the whole acceptor/T-domain is displaced by ~22 Å with the CCA-end positioned halfway between the 50S P and E sites (Fig. 1c,d). The tip of the triple helix bundle domain I of RRF, by interacting with the 23S rRNA P-loop, keeps the CCA-end of the tRNA in this position through shape complementarity (Fig. 1b). As a result, the base pairs that normally form between the CCA-end of the tRNA and the 23S rRNA P-loop are disrupted. The phosphate backbone of the CCA-end is constricted by the 23S rRNA helices H74 and H80, which block any further movement of the CCA-end (Fig. 3a,b and Extended Data Fig. 3). Nucleotides of the CCA-end are bunched together and packed against the phosphate backbone of helices H74 and H80 in the 50S subunit causing nucleotide A76 to flip by ~180°, suggesting that non-favorable interactions occur between the terminal end of the tRNA and the 23S rRNA (Fig. 3b and Extended Data Fig. 3).

Fig. 3 |. Interactions between the p/R-tRNA and the ribosome.

a, The p/R-tRNA (magenta) shown with its surrounding ribosomal elements. b, The phosphate backbone of the CCA-end of the p/R-tRNA is squeezed between the constriction formed by the 23S rRNA helices H74 and H80, blocking any further movement toward the E site. The protruding phosphate groups of the CCA-end (red) make non-favorable contacts with the phosphate backbone of helices H74 and H80. c, Several conserved basic residues of ribosomal protein uL5 interact with the TΨC loop (red) of the p/R-tRNA. d, In the p/R conformation, the deacylated tRNA in the P site moves away from the 23S rRNA helix H69. e, Conformational changes in the p/R-tRNA propagate to the ASL, weakening the base pairing interactions with the mRNA.

At the other end of the acceptor/T-domain is the elbow region of tRNA which, in this ribosome recycling complex, makes extensive interactions with ribosomal protein uL5. Two structured loops of uL5, composed of residues 46-50 (loop-1) and 74-85 (loop-2), form a unique flat surface with several basic residues facing outwards that extensively interact with the sugar-phosphate of nucleotides 50-56 of the TΨC loop of the tRNA (Figs 3c and 4a). The basic residues in uL5 are conserved from bacteria to human (Fig. 4d), suggesting that the interactions between uL5 and the TΨC loop of the tRNA are functionally important. In agreement with this premise, the cryo-EM reconstruction of the E. coli ribosome reported that during tRNA movement through the ribosome, the conserved loop-2 of uL5, referred to as the “P-site loop” ³⁸, interacts with tRNA as it moves from the P to the E site ²². Similar contact between rpL11 (uL5) in the mammalian 80S ribosome and the P-site tRNA was also reported ³⁹.

Fig. 4 |. Interactions between the TΨC loop of the p/R-tRNA and ribosomal protein uL5.

a, Displacement of the p/R-tRNA follows a smooth surface formed by two loops (green) in ribosomal protein uL5. The three conserved basic residues Lys81, Arg83, and Lys84 are shown. b, The apical G19:C56 base pair of the p/P-tRNA interacts with the loop-1 of uL5. c, In the recycling complex, the whole TΨC loop of the p/R-tRNA packs against the two loops of uL5 with Lys81 and Arg83 interacting with nucleotide bases in the major groove of the TΨC loop. d, The basic residues in loop-2 of protein uL5, also referred as the “P-site loop” ³⁸, are conserved from bacteria to human. Ribosomal protein uL5 is shown in yellow in the yeast 80S ribosome (PDB 6GQB ⁵⁵) and magenta in the human 80S ribosome (PDB 6EK0 ⁵⁶). Shown below is a structure-based sequence alignment of uL5 from Thermus Thermophilus (Tth), Escherichia coli (Eco), Saccharomyces cerevisiae (Sce), and Homo sapiens (Hsa). Only the region encompassing loop-1 and loop-2, boxed by red dashed lines, is displayed. Orange and light-blue highlights, conserved and semi-conserved residues, respectively. The conserved basic residues in loop-2 interacting with the p/R-tRNA are indicated by red stars.

The bend along the ASL of tRNA displaces the D-stem by ~12 Å toward the E site, breaking the interactions with the 23S rRNA helix H69 (Fig. 3d). Despite the conformational changes observed in the upper part of the tRNA, the anticodon remains essentially in the P site of the 30S subunit (Fig. 1c and Extended Data Fig. 4c). To our knowledge, this state of tRNA binding has not yet been reported and given its functional significance during ribosome recycling, we refer to it as the p/R- (peptidyl/recycling) tRNA.

The conformational changes in the p/R-tRNA propagate to the anticodon loop.

The RRF-mediated rearrangements in the p/R-tRNA propagate to the anticodon region located in the 30S P site, weakening the codon-anticodon base pair interactions (Fig. 3e). This is in contrast to the p/E hybrid state of a deacylated tRNA binding in transit between the P and E sites during tRNA translocation, for which the conformation of the anticodon remains essentially unchanged ^15,20. Upon building the model of the tRNA, we found that the electron density corresponding to the phosphate backbone of the ASL was poorly resolved and instead, clear electron density was observed for the nucleotide bases of the anticodon loop (Extended Data Fig. 4a). The final structure has high Wilson B-factor values for atoms of the phosphate backbone of the p/R-tRNA ASL (Extended Data Fig. 4b), suggesting that the ASL is in an unstable state of binding. Accordingly, the base pairs that normally take place between the anticodon and the P-site codon are virtually broken, with an average distance between non-bonding atoms of about 4 Å (Fig. 3e). The rearrangements of the ASL in the p/R-tRNA also cause a slight displacement of the ASL of the E-site tRNA by ~3 Å toward the exit site (Extended Data Fig. 4c). This agrees with the observation that the E-site tRNA only weakly interacts with the mRNA ⁴⁰.

RRF induces a conformation of the CCA-end of the p/R-tRNA that results in non-favorable phosphate-phosphate contacts with the 23S rRNA (Fig. 3b). Other parts of the p/R-tRNA are not compatible with the formation of canonical interactions with the 50S subunit ribosomal elements and the mRNA codon in the 30S P site. Our results provide a structural basis for how EF-G and RRF, with the deacylated tRNA in the P site, may promote disassembly of the ribosome into individual subunits. The structure supports data showing that despite the absence of tRNA translocation during ribosome recycling ^4,8, the P-site tRNA must be deacylated for the splitting reaction to occur rapidly ^8,16. Our study provides a basis for the observation that the interaction between RRF and a non-ratcheted ribosome in the post-translocation state with peptidyl-tRNA in the P site is weak ²¹, explaining why EF-G and RRF do not dissociate such ribosome complexes ⁸.

Conformational similarity between the p/R-tRNA and initiator tRNA.

It is striking how the conformation of the p/R-tRNA in this recycling complex resembles that of the p/I initiator tRNA that was described in both prokaryotic and eukaryotic initiation complexes (Fig. 5 and Extended Data Fig. 5) ^41–45. The p/R- and p/I-tRNAs occupy almost the same position except for their CCA-ends. The CCA-end of the p/I-tRNA is drawn toward the P site through its interaction with initiation factor 2 (IF2) ⁴⁴ or eukaryotic initiation factor 5B (eIF5B) ⁴², while that of the p/R-tRNA is kept away from the P site and against 23S rRNA helices H74 and H80 by RRF (Fig. 3b and Extended Data Fig. 3a). The compact conformation of EF-G, together with RRF in this ribosome recycling complex, constitute a molecular mimic of the overall shape of IF2 and eIF5B during initiation (Fig. 5 and Extended Data Fig. 5), thereby favoring binding of tRNA into analogous states. In the initiation complex, the proposed function of the p/I-tRNA conformation is to facilitate 50S subunit joining by allowing the 23S rRNA helix H69 to insert under the D-stem of the initiator tRNA and form one of the key inter-subunit bridges (B2a) with 16S rRNA helix h44 ⁴³. Similarly, the body of the p/R-tRNA, by adopting a conformation similar to that of the p/I-tRNA, disengages the 23S rRNA elements on the 50S subunit, setting the stage for dissociation of the 50S subunit. The non-favorable interactions between the CCA-end of the p/R-tRNA and the 23S rRNA helices H74 and H80 may facilitate splitting of the ribosome into subunits.

Fig. 5 |. Conformation of the p/R-tRNA compared to that of the p/I state during initiation.

Superimposition of the current recycling complex with that of an initiation 70S complex bound to the initiation factor 2 (IF2) (orange) (PDB 3JCJ ⁴⁴) reveals that conformation of the p/R-tRNA (magenta) resembles that of the p/I-tRNA (yellow). It also shows that the overall shape of RRF and EF-G mimics the shape of IF2, providing a missing link between translation initiation and ribosome recycling.

Discussion

This study shows how the deacylated tRNA exits the P site of the ribosome during recycling, which may lead to its transition to the E site or its complete dissociation from the ribosome as reported previously during recycling ^6,7,46,47. As the CCA-end of the p/R-tRNA transits from the P to the E site on the 50S subunit, the triple-helix bundle domain I of RRF maintains it in a position that leads to non-favorable phosphate-phosphate interactions with ribosomal RNA, which may result in tension that builds up between the tRNA and the 50S subunit. The weakening of the base pair interactions with the mRNA and the absence of interaction between the D-domain of tRNA and 23S rRNA helix H69 suggest that the p/R-tRNA is in an unstable state of binding. The conformation of the p/R-tRNA is strikingly similar to that of the p/I-tRNA in ribosome initiation complexes ^41–45, suggesting that ribosome disassembly into subunits follows the reverse order of events from that of initiation, and the conformation of tRNA bound in the p/R and p/I hybrid states is a key landmark in both processes.

Our structure provides details of RRF binding to the ribosome with its domain II positioned such that it simultaneously interacts with helix H69 in the 50S subunit, ribosomal protein uS12 and helix h44 in the 30S subunit (Fig. 2c). In all previous crystal structures of RRF bound to the ribosome in the absence of EF-G, domain II of RRF is flexible and faces outward into the inter-subunit space (Fig. 2b, Extended Data Fig. 2b, and Supplementary Video 1). In this ribosome recycling complex, EF-G extensively interacts with domain II of RRF, causing it to rotate toward the central inter-subunit bridge B2a wedging between H69 and uS12. This is consistent with low-resolution cryo-EM reconstructions of ribosome recycling complexes that reported large-scale rotation of domain II of RRF induced by EF-G ^10,11,35, and with biochemical data showing that RRF cooperates with EF-G to disassemble the ribosome ^3–9. It is expected that during recycling of the ribosome, EF-G would be in the GTP conformation with the ribosome in the fully ratcheted state. The transition of EF-G from the compact to the extended conformation would lead to further rotation of RRF domain II that would eventually break bridges B2a, and possibly B3, leading to dissociation of the ribosome into subunits. Notably, extensive rotation of the RRF domain II induced by the extended EF-G-GTP was observed in a low-resolution cryo-EM structure ³⁵.

The upper part of the p/R-tRNA, which comprises of the acceptor and T-domains, tightly fits between 23S rRNA helices H74 and H80 at the CCA-end, and ribosomal protein uL5 at the elbow region. Protein uL5 forms a flat surface composed of several conserved basic residues that interact with the elbow region of the p/R-tRNA (Figs 3c and 4a). The positively charged residues interact with the phosphate groups in the tRNA, suggesting the contact between uL5 and tRNA is universal and functionally important. In the classic p/P state, the apical G19:C56 base pair of the TΨC loop in tRNA forms a few interactions with the conserved residues Lys81, Arg83, and Lys84 in the loop-2 of uL5 ^40,48–50 (Fig. 4a,b). In this recycling complex, the elbow of the tRNA has slid over the loops of uL5 resulting in the close packing with uL5. Most of the close contact interactions occur between the backbone of nucleotides U50 to C56 of the tRNA and the two loops of uL5, with the basic residues Lys81 and Arg83 of uL5 inserting into the major groove of the TΨC loop of tRNA (Fig. 4a,c). Genetic studies in yeast have shown that mutations of the basic residues in the loop-2 region of uL5, composed of residues 74-85 (48-63 in yeast rpL11) and previously described as the “P-site loop”, lead to many defects including decreased fidelity of translation and increased frameshift ³⁸. Consistently, mutations in the TΨC loop of tRNA also cause frameshift and one proposed theory was that they weaken the interactions with uL5 ^51,52. A previous cryo-EM study of tRNA movement through the E. coli ribosome implicates the P-site loop of uL5 as a dynamic arm interacting with tRNAs as they transit between the P and E sites ²². Once tRNA takes the p/E hybrid position, it loses all interactions with uL5 (Fig. 6c). In the present complex, the p/R conformation of the deacylated tRNA allows to visualize the interactions mediated by the P-site loop of uL5, which guides the tRNA as it exits the P site.

Fig. 6 |. Implications for tRNA translocation on the 50S subunit during the elongation cycle.

a, Cross view of the 50S subunit through the tRNA binding sites. The canonical A, P, and E sites are indicated. The white dashed arrow shows the proposed trajectory of the tRNA CCA-end moving from the P to the E site during tRNA translocation. All tRNA models are from crystal structures of ribosome complexes aligned by the 23S rRNA. The PDB accession codes for these structures are 4WPO for the classical a/A- (brown) and p/P-tRNA (purple) ²⁵, 4V9C for the p/PE-tRNA (green) ⁵³, 4V9D for the p/E-tRNA (yellow) ¹⁵, and the p/R-tRNA is from this work. Note that the p/PE-tRNA (green) was originally named P/pe-tRNA ⁵³, however, to keep the consistency of nomenclature, we use p/PE instead as the ASL of the tRNA remains in the P site on the small 30S subunit. b, The elongated groove-like channel spanning from the A to the E site on the 50S subunit is indicated by a yellow dashed arrow. c, Ribosomal protein uL5 escorts the tRNA during its translocation from the P to the E site.

Extensive biochemical and structural studies have revealed most of tRNA trajectories on the ribosome, in particular how the anticodon stem of tRNA translocates on the small 30S subunit ^{18–20,22,23}. On the large 50S subunit, the P and E sites do not align horizontally; the E site is offset vertically from the P site by a distance of three nucleotides (Fig. 6a), owing to the 23S rRNA P-loop that base pairs with the CCA-end of the P-site tRNA. Movement of the tRNA terminal end from the P to the E site likely begins with a bend of the upper part of the tRNA detaching the CCA-end from the 23S rRNA P-loop, thus taking a similar configuration as the p/R-tRNA observed in our recycling complex. Instead of being trapped between 23S rRNA helices H74 and H80, further bending of the tRNA would effectively relieve the tension that builds up between the CCA-end and the 23S rRNA, such that it becomes unrestrained and moves laterally to the E site (Fig. 6a). Interestingly, a groove-like channel formed by 23S rRNA can be discerned on the 50S subunit between the P and E sites (Fig. 6b). Consistent with this prediction, in one previous crystal structure of a ribosome complex trapped by the antibiotic neomycin, the P-site tRNA adopts a hybrid state between the p/P and the p/E configurations ⁵³. The elbow region of the p/PE-tRNA is further twisted compared to the p/R-tRNA, which enables the CCA-end to reach the E site on the 50S subunit (Fig. 6a). The essential protein uL5 seems to play an indispensable role in stabilizing the tRNA and acting as a “guardrail” during its translocation from the P to the E site (Fig. 6c). The whole acceptor/T-domain of the tRNA is sandwiched between uL5 and the groove channel on the 50S subunit. The sliding motion of the TΨC loop along the flat surface of uL5 guides the tRNA as it travels between the P and E sites on the 50S subunit. Interestingly, molecular dynamics simulations of tRNA translocation proposed that the displacement of tRNAs is controlled by “sliding” and “stepping” mechanisms involving protein uL5 ⁵⁴.

During tRNA translocation, the A-site tRNA is bound in the a/P hybrid state following peptide bond formation ^18,23,24. The anticodon of the A-site tRNA interacts strongly with the 30S subunit by base pairing with the mRNA codon, whereas the peptidyl CCA-end establishes base pair interactions with the 23S rRNA P-loop on the 50S subunit. Thus, the peptidyl a/P-tRNA maintains the stable association between the two ribosomal subunits. This is in contrast to RRF during recycling that destabilizes critical inter-subunit contacts. In the absence of RRF during tRNA translocation, the CCA-end of the P-site tRNA remains unrestrained and will not be forced against the 23S rRNA helices H74 and H80. It will be transferred to the E site of the 50S subunit upon swiveling of the head domain of the 30S subunit, as observed in several structures upon tRNA binding in the p/E hybrid state ^15,18–20. The trajectory taken by the CCA-end may be relatively close to that seen here in the presence of RRF. Thus, we suggest that the body of the tRNA will take a similar positioning between the P and E sites during the EF-G-mediated tRNA translocation.

In summary, our structure provides insights into the mechanism of ribosome recycling by the concerted action of EF-G, RRF, and deacylated tRNA leading to subunit splitting. Binding of EF-G on the post-termination ribosome results in a conformation of RRF ready to attack the central inter-subunit bridges B2a and B3. The novel conformation of the P-site tRNA bound in the p/R hybrid state, which has eluded all previous observations, reveals new and likely transient interactions between the p/R-tRNA and the 50S subunit of the ribosome. Notably, the compaction of the CCA-end in the p/R-tRNA creates tension between the tRNA and the 50S subunit, suggesting a spring-like mechanism that would facilitate subunit splitting during recycling of the ribosome. This model provides a rationale for the reported increased rate of subunit splitting in the presence of deacylated tRNA ¹⁶. The disengagement of the p/R-tRNA from the mRNA and other 50S subunit elements may also facilitate its dissociation from the ribosome ^6,7,46,47. The visualization of a flat surface on protein uL5 that guides tRNAs as they transit between the P and E sites of the ribosome has implications for understanding the mechanism of tRNA movement on the ribosome.

Methods

Preparation of EF-G, RRF, tRNA^Phe and mRNA.

The full-length DNA sequence of the T. thermophilus EF-G protein fused at its N-termini to the DNA coding sequence for the first 74 amino acid residues of ribosomal protein bL9 was cloned into the pET28a plasmid (Novagen, Madison, WI) previously digested with NcoI and BamHI restriction enzymes. L9–EF-G gene fusion was incorporated into the pET28a plasmid using the sequence and ligation independent cloning. The L9(Asn74)-EF-G(Asp8) was previously used to obtain the crystal structure of the pre-translocation ribosome ²⁵. Expression was carried out as previously described by transforming E. coli BL21 (DE3) Star (Invitrogen, Carlsbad, CA) cells with the pET28a-L9(N74)-EF-G(D8) plasmid. The cells were grown in the LB medium in the presence of 30 μg/mL kanamycin (GoldBio, St-Louis, MO) to an absorbance of ~1.0–1.5 at 600 nm before inducing expression of the L9–EF-G protein fusion with 0.4 mM isopropylthiol-β-D-galactoside (IPTG, GoldBio, St-Louis, MO) for 3 hours at 37 °C. The cells were harvested, flash-frozen in liquid nitrogen, and stored at −80 °C. To purify the L9–EF-G fusion protein, approximatively 15 g of cell paste was lysed and treated as described previously ²⁵.

The RRF expression and purification were done essentially as previously described with minor modifications ¹⁴. The full-length RRF gene was PCR amplified from T. thermophilus HB8 genomic DNA and cloned into the pET30a (Novagen, Madison, WI) plasmid previously digested with NdeI and XhoI restriction enzymes, which adds a C-terminal hexa-histidine tag. The E. coli BL21 (DE3) pLysS was transformed with the construct and cultured in the LB medium in the presence of 30 μg/mL kanamycin to an absorbance of 0.5–0.7 at 600 nm before inducing RRF expression with 0.4 mM IPTG for 3 hours at 37 °C. Harvested cells were flash-frozen in liquid nitrogen and stored at −80°C. For purification of RRF, the frozen cell paste (15 g) with the overexpressed protein was re-suspended at 4 °C in buffer A (50 mM Tris-HCl pH 7.5, 200 mM KCl, 1 mM β-mercaptoethanol) with one complete EDTA-free protease inhibitor tablet. The re-suspended cells were disrupted by three passes through a microfluidizer (15,000 PSI, Microfluidics, Newton, MA). The cell debris were removed by centrifugation at 18,000 rpm at 4 °C for 45 minutes. The supernatant was heated at 65 °C for 20 minutes and centrifuged at 10,000 rpm at 4 °C for 45 minutes to remove denatured proteins from the expression host. Clarified supernatant was filtered through a 0.22-μm filter (Millipore, Burlington, MA), loaded onto one 5 mL nickel HisTrap HP (GE Healthcare, Piscataway, NJ) fast-flow column previously equilibrated in buffer A. Elution of RRF from the HisTrap column was performed by a linear gradient of buffer A containing 500 mM imidazole. The fractions containing RRF were combined, concentrated and precipitated by adding 3 M ammonium sulfate and retained on ice for 1 hour with slow agitation. After centrifugation at 6,000 rpm at 4 °C for 30 minutes, the pellet was resuspended in buffer G (20 mM MES-KOH pH 6.0, 200 mM KCl, 1 mM β-mercaptoethanol). The sample was filtered through a 0.22-μm filter and loaded onto a size exclusion chromatography column (Superdex 75 26/60, GE Healthcare, Piscataway, NJ). The peak fractions containing RRF were pooled, and concentrated to approximatively 90 mg ml⁻¹. The RRF was flash-frozen in liquid nitrogen in small aliquots and stored at −80 °C until used in crystallization experiments.

The 21-mer mRNA, with a Shine-Dalgarno sequence, had the sequence 5’ C AAG GAG GAA AAA UUC UAA UA 3’ with the UUC codon in the P site, and the UAA stop codon in the A site (underlined) was chemically synthesized by Integrated DNA Technologies (Coralville, IA). The E. coli tRNA^Phe was expressed and purified as previously described ⁵⁷.

Formation of the 70S ribosome recycling complex.

The ribosome complex was formed as previously reported with modifications ²⁵. We used 70S ribosomes isolated from a mutant strain of T. thermophilus HB8 that carries a truncated ribosomal protein bL9 in its genome ^25,31. Because these ribosomes lack ribosomal protein bL9 altogether, we replaced it with the L9–EF-G fusion during crystallization. First, 4 μM 70S:L9₅₈ ribosomes were incubated with 8 μM mRNA in buffer G (5 mM HEPES-KOH pH 7.5, 10 mM Mg(CH₃COO)₂, 50 mM KCl and 10 mM NH₄Cl, and 6 mM β-mercaptoethanol) at 55 °C for 5 minutes. Following addition of 30 μM RRF, the mixture was incubated at room temperature for 15 minutes. Guanosine 5’-diphosphate (GDP) nucleotide was added to a final concentration of 50 μM, together with 5 μM L9–EF-G, and incubated 15 minutes at room temperature. The deacylated tRNA^Phe was added to a final concentration of 20 μM and the complex incubated at room temperature for 15 more minutes. The higher than usual tRNA concentration used in previous studies ^25,31,58 during complex formation allowed trapping a pre-recycling ribosome with RRF, EF-G, and two tRNA^Phe bound simultaneously. Finally, the complex was then allowed to reach equilibrium at room temperature for 10 minutes prior to use in crystallization experiments.

Crystallization.

Crystals were grown at room temperature in sitting drop trays in which 3 μL of ribosome complex was mixed with 4 μL reservoir solution containing 100 mM Tris-HCl (pH 7.3), 2.4–2.7% (w/v) polyethylene glycol (PEG) 20,000, 7–9% (v/v) 2-methyl-2,4-pentanediol (MPD), 150 mM L-arginine-HCl, and 0.5 mM β-mercaptoethanol. Ribosome crystals grew to full size within 5–7 days. The crystals were transferred stepwise into cryo-protectant solution with increasing MPD concentrations and containing 100 mM Tris-HCl pH 7.3, 10 mM Mg(CH₃COO)₂, 50 mM KCl, 10 mM NH₄Cl, 6 mM β-mercaptoethanol, 2.7% (w/v) PEG 20,000, and 100 μM RRF, in which they were incubated overnight at room temperature. After stabilization, crystals of the ribosome recycling complex were harvested and immediately frozen in a nitrogen cryostream at 80 K before being plunged into liquid nitrogen.

X-ray data collection and structure determination.

X-ray diffraction data were collected at beamline 24-ID-C at the Advanced Photon Source at Argonne National Laboratory (Argonne, IL), and beamline BL17U at the Shanghai Synchrotron Radiation Facility (Shanghai, China). The final complete data set of the 70S ribosome recycling complex used in this study was collected from a single crystal at 100 K with 0.3º oscillations and 0.9791 Å wavelength on beamline 24-ID-C at the Advanced Photon Source at Argonne National Laboratory (Argonne, IL). The raw data were integrated and scaled with the XDS program package ⁵⁹. The complex of the ribosome with EF-G, GDP, RRF, mRNA, and tRNA^Phe crystallized in the primitive orthorhombic space group P2₁2₁2₁ with approximate unit cell dimensions of 210 Å by 448 Å by 624 Å and contained two ribosomes per asymmetric unit of the crystal (Table 1). The structure was solved by molecular replacement with PHASER from the CCP4 suite ⁶⁰. The search model was generated from the published high-resolution structure of the T. thermophilus 70S ribosome with all ligands removed (PDB 4Y4O ³³). The initial molecular replacement solution was refined by rigid-body refinement with the ribosome split into multiple domains, followed by five cycles of positional and individual B-factor refinement with PHENIX ⁶¹. After initial refinement, there was clear electron density corresponding to the L9–EF-G, RRF, mRNA and two tRNAs in the P and E sites in the unbiased F_o – F_c difference Fourier maps.

Model building and refinement.

L9–EF-G, RRF, mRNA, p/R-tRNA, and E-tRNA were built into the unbiased difference density map from the initial round of refinement, and the refinement scheme described above was performed after addition of each ligand. The final model of the ribosome recycling complex was generated by multiple rounds of model building in COOT ⁶² and subsequent refinement in PHENIX ⁶¹. The statistics of data collection and refinement for the complex are compiled in Table 1. All figures were generated with PyMOL ⁶³.

Data availability.

Atomic coordinates and structure factors have been deposited in the Protein Data Bank under the accession code 6UCQ.

Extended Data

Extended Data Fig. 1 |. Difference Fourier map of the 70S ribosome recycling complex and conformation of the ribosome.

a, Unbiased F_o – F_c difference electron density map contoured at 2.0 σ following the first round of refinement. Both deacylated tRNAs in the P and E sites, together with the mRNA, RRF, and EF-G were clearly visible. b, In the 70S ribosome recycling complex (orange), the 30S subunit rotates counter-clockwise by ~2° compared to the classical state ribosome (blue).

Extended Data Fig. 2 |. Conformation of RRF on the ribosome in the recycling complex.

a, Interactions between domain I of RRF and the ribosomal RNA. Shown are parts of H74 and H80 of the 23S rRNA (white), the CCA-end of the p/R-tRNA (pink), and domain I of RRF (blue). Key interacting residues in RRF are shown. b, Compared with the E. coli 70S ribosome bound to RRF (PDB 4V54¹³), domain II of RRF in the current structure rotates by ~28° and becomes wedged between the 23S rRNA helix H69 (orange) and ribosomal protein uS12 (magenta).

Extended Data Fig. 3 |. Difference Fourier map of the CCA-end of the p/R-tRNA.

a, Unbiased F_o – F_c map contoured at 2.0 σ of the 3’ end of the p/R-tRNA showing its location relative to the 23S rRNA helices H74 and H80. b, Rotated view of panel A with ribosomal elements omitted for clarity.

Extended Data Fig. 4 |. Conformation of anticodon stem loop (ASL) of tRNAs in the recycling complex.

a, Unstable state of binding of the p/R-tRNA ASL in the P site of the ribosome recycling complex. The unbiased F_o – F_c difference Fourier map contoured at 2.0 σ of the ASL of the p/R-tRNA shows that the electron density is much clearer for the nucleotide bases than for the phosphate backbone. b, Average Wilson B-factor values for the phosphate backbone (blue bars) and the nucleotide bases (orange bars) of the p/R-tRNA ASL. c, Movements of ASLs in the P and E site in the recycling complex compared to tRNAs bound in the classical state (gray).

Extended Data Fig. 5 |. Conformation of the p/R-tRNA compared to that of the p/I state during initiation.

Superimposition of the current recycling complex with that of a eukaryotic initiation 80S complex bound to eIF5B (orange) (PDB 4V8Z⁴²) shows that like the IF2-70S initiation complex (Fig. 5), that the conformation of the p/R-tRNA (magenta) resembles that of the p/I-tRNA (yellow). The same molecular mimic is observed between RRF/EF-G and eIF5B as that seen with IF2.

Supplementary Material

Supplementary Video 1

Supplementary Video 1 | Structure of the bacterial ribosome recycling complex. Animation showing the interactions between EF-G and RRF with the ribosome and the p/R-tRNA. The conformation of tRNAs in the recycling complex is compared with that of tRNAs bound in the canonical e/E and p/P states. The position of domain II of RRF is compared to a previous ribosome-bound RRF structure in the absence of EF-G (PDB 4V54¹³). The RRF-mediated displacement of the P-site tRNA from the p/P to the p/R state is guided by ribosomal protein uL5 shown in green.