Experimental research paper/ Introduction&background part

The ribosome, which constitutes one of the most com­ plex and sophisticated macromolecules in the bacterial cell, lies at the centre of translation. In bacteria, the small 30S ribosomal subunit associates with the large 50S sub­ unit to form a functional 70S ribosome. The 30S subunit consists of the 16S ribosomal RNA (rRNA) and 21 pro­ teins (denoted S1–S21; prefix S for ‘small’), whereas the 50S subunit contains two rRNAs (the 23S and 5S rRNAs) and 33 different proteins (known as L proteins; prefix L for ‘large’)1. All components are present in one copy, with the exception of L7/L12, which is present in four or six copies per ribosome in bacteria2,3 and archaea4,5 (L7 is the N­acetylated form of L12). These proteins are the only ribosomal proteins that do not directly interact with rRNA; their binding is mediated by L10, and together they form a stable pentameric or heptameric complex 6 known as the L7/L12 stalk (referred to hereafter as the L12 stalk). This stalk is an essential component of the docking site for the translational guanosine­nucleotide­binding proteins (G proteins), which assist the ribosome at vari­ ous stages of translation. Despite the large number of ribosomal proteins, rRNA is the dominant component in terms of both structure and function (FIG. 1). Decoding of the mRNA is carried out by elements of the 16S rRNA7,8, and peptide­bond formation is carried out by nucleotides of the 23S rRNA9–11 (reviewed in REF. 12). Ribosomal proteins have important roles in ribosome biogenesis13,14, in maintaining the overall architecture of the rRNA, and they have also been implicated in a num­ ber of important functional activities, including mRNA

helicase activity (for S3, S4 and S5)15, decoding (for S12)7 and peptidyltransferase activity (for L27 (REF. 16) and L2 (REF. 17)).

The ribosome passes through four functional phases for the synthesis of a single protein: initiation, elonga­ tion, termination and recycling (FIG. 2). All phases are mediated by specific factors, some of which are bacteria­ specific, whereas others (such as the elongation factors EF­Tu and EF­G) are universally conserved. The amino acid substrates that are attached to tRNAs (known as aminoacyl­tRNAs (aa­tRNAs)) are delivered to the ribo­ some in a ternary complex with EF­Tu and GTP, and the tRNAs move through three distinct binding sites (the aminoacyl­ (A­), peptidyl­ (P­) and exit­ (E­) sites) located at the interface of the 30S and 50S subunits.

After initiation — which involves placement of the mRNA start codon and the specific initiator tRNA (formyl methionine tRNA; fMet­tRNA) at the P­site of the 30S subunit, followed by association of the 50S sub unit — the elongation cycle ensues. The ribo­ some moves along an mRNA in the 5ʹ to 3ʹ direction and decodes each consecutive codon with the help of the incoming aa­tRNAs. After successful decoding, the aa­tRNA swings fully into the A­site (in a process that is known as accommodation). Decoding and accommo­ dation are often collectively referred to as ‘A­site occu­ pation’. The swing docks the aminoacyl residue into the peptidyltransferase centre, resulting in rapid peptide bond formation. The nascent chain is transferred from the peptidyl­tRNA at the P­site to the charged tRNA at

Decoding Selection of the cognate ternary complex of aminoacyl- tRNA–EF-Tu–GTP on the basis of correct codon-anticodon interactions between the mRNA and tRNA, respectively.

EF‑G and EF4: translocation and back‑translocation on the bacterial ribosome Hiroshi Yamamoto1*, Yan Qin2*, John Achenbach3*, Chengmin Li2, Jaroslaw Kijek4, Christian M. T. Spahn1 and Knud H. Nierhaus1,4

Abstract | Ribosomes translate the codon sequence of an mRNA into the amino acid sequence of the corresponding protein. One of the most crucial events is the translocation reaction, which involves movement of both the mRNA and the attached tRNAs by one codon length and is catalysed by the GTPase elongation factor G (EF‑G). Interestingly, recent studies have identified a structurally related GTPase, EF4, that catalyses movement of the tRNA

2 –mRNA complex in the opposite direction when the ribosome stalls, which is known as

back‑translocation. In this Review, we describe recent insights into the mechanistic basis of both translocation and back‑translocation.

1Institut für Medizinische Physik und Biophysik, Charité – Universitätsmedizin Berlin, Charitéplatz 1,10117 Berlin, Germany. 2Laboratory of noncoding RNA, Institute of Biophysics, Chinese Academy of Science; 15 Datun Road, Beijing 100101, China. 3NOXXON Pharma AG, Max-Dohrn-Strasse 8–10, 10589 Berlin, Germany. 4Max Planck Institut für molekulare Genetik, Ihnestrasse 73, D-14195 Berlin, Germany. *These authors contributed equally to this work. Correspondence to K.H.N.  e-mail: nierhaus@molgen. mpg.de doi:10.1038/nrmicro3176 Published online 23 December 2013

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the A­site, thus deacylating the P­site tRNA and extend­ ing the nascent chain by one amino acid. The tRNAs must then be moved in a step known as translocation. In this Review, we classify all tRNA conformational states after peptide bond formation and before translocation as pre­translocational states (PRE­states). To accom­ modate the next incoming aa­tRNA, the peptidyl­tRNA at the A­site and the deacylated tRNA at the P­site are translocated to the P­ and E­sites, respectively, and this is catalysed by EF­G–GTP. The resulting state, in which the P­ and E­sites are occupied and the A­site is vacant, is called the post­translocational state (POST­state) (reviewed in REF. 18). The release of the deacylated tRNA from the E­site is thought to occur after trans location19,20 or, alternatively, on occupation of the A­site with the next aa­tRNA21–23.

It is possible that ribosomes mistranslocate, which leads to an arrest in protein synthesis as the ribosome stalls and thereby blocks the progression of other ribo­ somes on the same mRNA. Recent studies suggest that such stalled ribosomes can be rescued by a GTPase known as EF4, which is structurally related to EF­G. This factor recognizes stalled ribosomes that have a deacylated tRNA in the E­site and a peptidyl­tRNA in the P­site (the POST­state) and catalyses a back­translocation reaction (FIG. 2). The tRNAs are dragged back into the P­ and A­sites, thereby giving the ribosome a second chance to properly translocate24–26. Other studies suggest that EF4 can also bind to and mobilize ribosomes that are stalled in the PRE­state27 (see below). Translation is

terminated when a ribosome encounters a stop codon on the mRNA, which is recognized by a release factor that triggers release of the nascent polypeptide. During the final phase of translation, which is known as recycling, the 70S ribosome is thought to dissociate into its 30S and 50S subunits, which are re­used for subsequent rounds of initiation (reviewed in REF. 18).

In this Review, we discuss a number of recent struc­ tural and biochemical studies in bacteria, primarily Escherichia coli and Thermus thermophilus, that have enhanced our understanding of the mechanisms of bac­ terial translocation and back­translocation. The binding modes and functional roles of EF­G and EF4 are dis­ cussed, as well as the proposed physiological relevance of back­translocation.

EF‑G and EF4 Structural similarities. EF­G and EF­Tu are universal translation factors, whereas EF4 is found in almost all bacteria, in mitochondria and chloroplasts, but is absent in archaea and the cytoplasm of eukaryotes. EF4 is the third most highly conserved bacterial protein after EF­Tu and EF­G, with a 55–68% amino acid identity between different bacterial species24.

The three­dimensional structures of EF­G and the ternary complex (aa­tRNA–EF­Tu–GTP) are highly similar (FIG. 3a,b). The five structural domains of EF­G (FIG. 3a) fold into a structure that resembles the ternary complex, and domain IV of EF­G corresponds to the anticodon stem–loop of the tRNA within the ternary

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50S subunit

L12

L12 stalk

L1

L1 stalk

CP

PTC

Head

Body

Platform

S13

S12E P A

30S subunit

E P

A

L11 L10

SRL

23S rRNA 16S rRNA

5S rRNA

Figure 1 | Overall architecture of the large and small subunits of the bacterial ribosome. Both subunits are shown from the interface side. The large 50S subunit contains the 23S ribosomal RNA (rRNA) and 5S rRNA (light grey and dark grey, respectively), and the small 30S subunit is composed of the 16S rRNA (light grey). Ribosomal proteins are represented as coloured ribbons, and those that have specific roles in translocation, as well as the sarcin–ricin loop (SRL) of the 23S rRNA and the acceptor ends of A‑ and P‑site tRNAs within the peptidyl‑transferase centre (PTC), are highlighted by surface representation. The A‑site, P‑site and E‑site tRNAs are also shown. For clarity, only the anticodon stem‑loops of the tRNAs are shown on the 30S subunit. The structures were produced using coordinates from Protein Data Bank accessions 2WRL31, 2QA4 (REF. 112), 3A1Y5, 1RQU113 and 3J0T (50S subunit), and 2WRK31 and 3J0U46 (30S subunit). CP, central protuberance.

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Single‑turnover experiments Experiments in which the conditions are set such that the catalyst (for example, the ribosome) only undergoes a single round of catalysis.

complex (FIG. 3b). This is probably the most famous example of molecular mimicry, which highlights the need for both EF­G and the ternary complex to occupy a similar site at the interface of the ribosomal subunits. Similarly, the domain structure of EF4 is highly related to that of EF­G (FIG. 3a). Both factors share domains I (known as the G domain), II, III and V, which are responsible for ribosome binding and GTPase activ­ ity. In addition, both factors have specific domains: EF­G contains Gʹ (which is a sub­domain of domain I)

and domain IV, whereas EF4 has a unique carboxy­ terminal domain (CTD)24. Domain IV of EF­G and the CTD of EF4 are responsible for mediating the opposing roles of these two factors in translation (FIG. 3c).

First contacts with the ribosome. The first contacts of EF­G and EF4 with the ribosome involve the L12 stalk and seem to follow the same pathway. The substrate for EF­G is the 70S ribosome in the PRE­state, whereas the substrate for EF4 is still unclear. One study suggests that EF4 preferentially binds to the POST­state ribosome, owing to observations that EF4 binds to the POST­state with higher affinity than to the PRE­state, and that EF4­dependent GTP hydrolysis has a higher turnover rate with POST­state ribosomes than with PRE­state ribosomes28. However, single-turnover experiments and single-molecule FRET (Förster resonance energy trans­ fer) measurements suggest that the PRE­state is the preferential but not the exclusive target of EF4. In this study, EF4 could compete with EF­G for binding to the PRE­state27. Thus, EF­G recognizes a specific functional state, whereas EF4 seems to be more promiscuous in its specificity.

It is thought that EF­G makes its first ribosomal con­ tact with the CTD of L12 using the Gʹ domain3. The next step might be shared by other factors (such as EF­Tu and EF4) and involves contact with the base of the L12 stalk, resulting in interactions between the L12 CTD and the amino­terminal domain (NTD) of L11, as demonstrated by cryo­electron microscopy(cryo­EM)29,30 and X­ray crystallography31,32. This interaction is controlled by the universally conserved Pro22 residue of L11, which is in a trans­configuration when the ribosome is free of GTP­ binding proteins or when a non­GTPase factor is bound (Supplementary information S1 (figure)). However, when a G­protein factor such as EF­G, EF­Tu or EF4 binds to the ribosome, Pro22 adopts the cis­configuration, which facilitates the L11–L12 interaction. Interestingly, the trans–cis transition is catalysed by a peptidyl-prolyl cis–trans isomerase (PPIase) centre, comprising amino acyl residues that reside mainly in the G domain of translational fac­ tors. Before the factor dissociates from the ribosome after GTP hydrolysis and inorganic phosphate (Pi) release, the PPIase activity of the factor stimulates reversion of Pro22 to the trans­configuration33,34.

The early contacts of EF­G with the ribosome pre­ sent a conundrum: EF­G triggers the movement of the tRNA2–mRNA complex from a PRE­state to the POST­ state, but the initial EF­G contacts with the ribosome that are essential for activating the ribosome and setting the tRNA2–mRNA complex in motion are currently unknown. When EF­G is added to a PRE­state ribo­ some and its dissociation from the ribosome is inhibited (using the antibiotic fusidic acid or the non­cleavable GTP analogues GDPNP (guanosine 5ʹ­tetrahydro­ gen triphosphate) or GDPCP (5ʹ­guanosyl­methylene triphosphate), X­ray and cryo­EM structures have dem­ onstrated that the peptidyl­tRNA has left the A­site and approaches the P­site, and domain IV of EF­G is flipped into the A­site, where it functions as a doorstop to prevent back­translocation of the tRNA2–mRNA

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aa-tRNA–EF-Tu–GTP

EF-Tu–GDP + P i

A-site occupationTranslocation

Peptidyl transfer

EF-G–GTP

E-tRNA

EF-G–GDP + P i

Elongation cycle

Initiation

Termination

Recycling 70S

initiation complex

mRNA

fMet-tRNA

50S

30S APE

APE

EF4–GDP + P i

EF4–GTP

APE

APE

APE APE

Figure 2 | The functional phases of the ribosome during translation. The 70S initiation complex contains the initiator tRNA (formylmethionine tRNA (fMet‑tRNA)) at the ribosomal P‑site, which interacts with the start codon (typically AUG) of the mRNA via the formation of a codon–anticodon duplex. The 70S initiation complex enters the elongation cycle on binding the ternary complex aminoacyl‑tRNA–elongation factor Tu–GTP (aa‑tRNA–EF‑Tu–GTP). After successful decoding, GTP is hydrolysed, EF‑Tu–GDP and inorganic phosphate (P

i ) leaves the ribosome, and the aa‑tRNA swings into the A‑site

(A‑site occupation). The nascent peptide chain is transferred from the peptidyl‑tRNA in the P‑site to the aa‑tRNA in the A‑site, extending the peptide chain by one amino acid, in a reaction known as peptidyl transfer. Facilitated by EF‑G–GTP, the tRNA

2 –mRNA

complex is translocated by a distance of one codon from the A‑ and P‑sites to the P‑ and E‑sites. EF4–GTP can catalyse a reversal of this step, termed back‑translocation, in order to mobilize stalled ribosomes (dashed arrows). When a stop codon enters the A‑site, termination of protein synthesis occurs, which is assisted by release factors. The ribosome can now enter the recycling phase, after which a 70S initiation complex is formed again.

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Single‑molecule FRET

(Single-molecule Förster resonance energy transfer). A phenomenon in which energy induced by light excitation is transferred from one fluorophore to another in a distance-dependent manner, observed on a single complex or molecule.

complex 31,35–39 (FIG. 3c; Supplementary information S2 (figure)). In other words, in all previous ribosome struc­ tures with EF­G, the factor has already triggered a first step of translocation. However, a recent report describes the structure of a pre­translocational EF­G—ribosome complex with two tRNAs in hybrid positions. The com­ plex was prepared in the presence of GTP; EF­G disso­ ciation was blocked with the antibiotic fusidic acid and translocation of the tRNA2–mRNA complex was inhib­ ited with the antibiotic viomycin115. In this PRE­state, the

tip of EF­G domain IV makes strong contacts with the anticodon loop of the A­site tRNA. A comparison of the EF­G structure in the POST state31 revealed that EF­G undergoes a ~20° rotation around the sarcin–ricin loop (SRL) of the 23S rRNA. This rotation results in a movement of the tip of domain IV by 20 Å into the decoding centre during the transition from the PRE­ to the POST­state. Although this study reveals important insights, it is still unclear what triggers the dramatic conformational change of EF­G and which contacts between EF­G and the ribosome (or its ligands) set the tRNA2­mRNA in motion.

When EF4 is added to POST­state ribosomes, the structures that are available show the peptidyl­tRNA in a back­translocated position, having established either an intermediate state (possibly identical with a trans­ location intermediate25) or a PRE­state28. Thus, a struc­ ture in which EF4 is bound to the POST­state before the onset of back­translocation is currently lacking.

The specific domains of EF‑G and EF4. Both factors reduce the activation-energy barrier between PRE­ and POST­states, but the binding of each factor induces one distinct state of the tRNA2–mRNA complex; EF­G favours the POST­state and EF4 favours the PRE­state. EF­G flips domain IV into the A­site, resulting in a door­ stop effect that stabilizes the POST­state. This suggests that domain IV is essential for translocation. Indeed, Thermus thermophilus EF­G fragments that lack this domain are unable to translocate, but they retain GTPase activity and are able to bind to the ribosome40. As men­ tioned above, EF4 lacks domain IV of EF­G and, as such, lacks the doorstop function, which is considered to be a prerequisite to allow for the back­movement of tRNAs from the POST­state to the PRE­state. This is clearly seen in the cryo­EM structure28 (Supplementary information S2 (figure), left panel), in which the back­translocated peptidyl­tRNA in the A­site is attached to the unique CTD of EF4, whereas domain IV of EF­G would prevent movement into this position. After movement back into the A­site, the CTD of EF4 halts the peptidyl­tRNA in this position, thereby re­establishing the PRE­state. This halting effect is caused by surface patches of strong posi­ tive charges on EF4 that attract the negative charges of the A­site tRNA28,41. The CTD of EF4 contacts the inner side of the elbow and the acceptor­stem down to the CCA end of the A­site tRNA (Supplementary information S2 (figure), right panel).

To preserve the reading frame during back­translocation, maintenance of codon–anticodon interactions is essen­ tial. The presence of a cognate E­site tRNA is crucial for EF4­mediated back­translocation24 because a back­ translocated tRNA in the P­site must sustain codon– anticodon interactions; without such interactions, a P­site tRNA cannot be fixed on the 30S subunit42.

Mechanism of translocation A wealth of recent structural data describing the dynam­ ics and structural transitions of the ribosome during translocation now allows for a comprehensive overview of the mechanisms involved. In this section, we describe

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EF4

a

b

P/P

P/P

E/E

A/L

EF4

EF-G

EF-Tu

G Gʹ G II III IV V CTD

c

1–158 159–253 254–289 290–404 405–482 483–603

1– –212 213–313 314–405 tRNA

604–691

1– –188 189–281 291–371 398–486 487–599

EF-G

EF4

G

IIIII VV

CTD

EF-Tu

G

II III

A/T-tRNA

EF-G

G

II

III

IV

Gʹ

Common domainsSpecific domains

Backwards

Forwards

Figure 3 | Structure, binding sites and functions of the elongation factors. a | Domain organization of elongation factor G (EF‑G), EF4 and EF‑Tu. b | EF‑G, EF4 and EF‑Tu have a highly similar domain organization and fold into similar three‑dimensional structures (EF‑G, Protein Data Bank (PDB) accession 2WRI31; EF4, PDB accession 3DEG28; and the ternary complex aminoacyl‑tRNA−EF‑Tu−GTP, PDB accession 2WRN70). c | EF‑G and EF4 bind to a similar site on the ribosome, but their specific domains promote opposing effects. EF‑G catalyses forward movement of the tRNAs from the A/A and P/P sites to the P/P and E/E sites, whereas EF4 can reverse this reaction to promote back translocation, moving the tRNAs from E/E to P/P and from P/P even beyond the A/A site toward the L12 stalk. The latter position is only seen in the presence of EF4 and is referred to as the A/L position.

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Peptidyl‑prolyl cis–trans isomerase An enzyme that belongs to the peptidyl-prolyl isomerase (PPIase) family that catalyses the transition of a proline residue between cis and trans conformations by reducing the activation-energy barrier that separates these two conformations.

Sarcin–ricin loop (SRL). The loop of helix H95 (G2654–A2665; E. coli nomenclature), which contains the longest universally conserved ribosomal RNA (rRNA) sequence. Its name derives from the observations that removing base A2660 by the N-glycosidase ricin or cleaving the 23S rRNA after G2661 by the RNase α-sarcin impairs the binding and GTPase activity of both elongation factor Tu (EF-Tu) and EF-G, thereby blocking translation.

Activation‑energy barrier The energy barrier that separates reactants and products in a chemical reaction.

the role of intersubunit rotation (formerly called ‘ratch­ eting’43) and swivelling of the head of the 30S subunit in translocation, as well as recent insights into the role of GTP hydrolysis.

The PRE‑states. After peptide­bond formation, the ribo­ some can adopt at least three PRE­states; in each state, both the A­ and P­sites on the 30S subunit are occupied by a tRNA­anticodon stem, whereas the CCA ends of the tRNAs on the 50S subunit can vary in their location. In the classical PRE­state, the anticodon stem and the CCA end of the two tRNAs are positioned in the same site on each ribosomal subunit (known as A/A for the A­site tRNA and P/P for the P­site tRNA). The ribosome spontaneously fluctuates between this classical state and a rotated state44. Rotation involves a 4–7 ° anticlockwise rotation of the 30S subunit relative to the 50S subunit, around a pivot axis close to the middle of helix 44 (h44)43 (FIG. 4a). The intersubunit rotation is coupled to a move­ ment of the CCA end of the P­site tRNA on the 50S sub­ unit to the E­site; simultaneous movement of the CCA end of the A­site tRNA into the 50S P­site may occur

but is not strictly coupled. The tRNA positions within the 30S subunit remain unchanged, giving rise to hybrid sites45. The functional state of a ribosome with a tRNA in an A/P hybrid site (anticodon stem in the A­site on the 30S subunit and the CCA end in the P­site on the 50S subunit), and a deacylated tRNA in a P/E hybrid site (anticodon stem in the P­site of the 30S and the CCA end in the E­site of the 50S) is known as hybrid state 1 (H1). The third PRE­state (A/A and P/E), which corre­ sponds to movement of the P­site tRNA only, is known as hybrid state 2 (H2)44,46 (FIG. 4b). Back­rotation of the 30S subunit re­establishes the tRNAs in the classical A/A and P/P binding positions.

These fluctuations between the various PRE­states only occur in the absence of EF­G47. All three PRE­ states are substrates for EF­G; EF­G can enter the sequence of PRE­states (classical, H2 and H1) at any stage in order to move the tRNA2–mRNA complex to the POST­state, although EF­G–GTP seems to favour the 30S rotated state with tRNAs in hybrid positions48,49. In other words, this sequence of PRE­states is the only route to the transition state and is thus essential

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4–7°

Non-rotated

30S head

30S head

Rotated

Classical

H1 H2

18°

P/P A/A

P/E A/P P/E A/A

30S body

50S subunit

L1 stalk

L12 stalk

L12 stalk

L1 stalk

a

b

Swivelled

Non-swivelled

c

30S body

50S subunit

Figure 4 | The three PRE-states of tRNAs on the ribosome during translocation. a | Intersubunit rotation of the 30S subunit, viewed from the 30S solvent side with the 50S subunit in a fixed position. Rotation of the 30S subunit occurs in an anticlockwise direction by 4–7 ° and does not depend on elongation factor G (EF‑G). b | After peptidyl transfer, the tRNAs can shift between classical and hybrid states. In the classical pre‑translocational state (PRE‑state) the tRNAs are located in A/A and P/P positions, in the post‑translocational state (POST‑state), the tRNA adopts the P/P and E/E positions. However, in hybrid state 1 (H1), the tRNAs occupy the A/P and P/E positions and in hybrid state 2 (H2), they are located in the A/A and P/E positions. c | The second major conformational change that the 30S undergoes during translocation is termed swivelling. This movement is EF‑G‑dependent and involves an anticlockwise rotation of the 30S head towards the E‑site, which opens the A790 gate and moves the tRNA

2 –mRNA complex to the POST‑state.

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Polysomes mRNAs to which more than one ribosome is bound.

for translocation47. Inhibition of intersubunit rotation by crosslinking the 30S and 50S subunits blocks trans­ location50, which shows that this is an essential step in translocation. Single­molecule FRET measurements have revealed that there are two populations of pre­translocation complexes: one in which the ribosome rapidly fluctu­ ates between classical and hybrid states, and another in which the tRNA positions are long­lived in either the classical or hybrid state configuration. Following the addition of EF­G, both populations of pre­translocation complexes are translocated47, but it is currently unclear whether only one or both populations exist in vivo.

The transition from PRE‑states to the POST‑state. After binding to the A­site, a tRNA must translocate twice (from the A­site to the P­site and from the P­site to the E­site) during the course of translation, which involves five distinct combinations of tRNA binding sites: A/A, A/P, P/P, P/E and E/E. Analyses of ribosomes in polysomes51,52 or during poly(Phe) synthesis53 have revealed that at least two tRNAs are always present on the ribosome during the elongation cycle; in the PRE­state this corresponds to either the classical state (A/A and P/P) or the hybrid states (H1 or H2). By con­ trast, only one POST­state exists, which is characterized by a peptidyl­tRNA in the P/P site and a deacylated tRNA in the E/E site (Supplementary information S3 (figure)). A transition intermediate between the PRE­ and POST­ states is observed when EF­G is trapped on the ribosome either by using GDPNP or fusidic acid. This intermedi­ ate is characterized by another large­scale movement of the ribosome, this time exclusively within the small sub­ unit. It involves an anticlockwise rotation of the 30S head relative to the 30S body, termed swivelling, which turns the head by about 18 ° towards the E­site35–39,54–56 (FIG. 4c). In agreement with measurements of head rotation and mRNA movement 57, structural data show an almost complete translocation of the tRNA2–mRNA complex in the POST­state transition intermediate (TIPOST)35,58. EF­G dependent GTP hydrolysis is not required for translo­ cation, however, it must occur to ensure that EF­G is released from the ribosome. A reversal of the head swivel and 30S back­rotation ensues, thereby establishing the stable POST­state, in which the tRNAs fully occupy the P/P and E/E sites.

It is important to note that during translocation of the tRNA2–mRNA complex, it is the tRNAs that are physically moved by the ribosome, whereas the mRNA co­migrates with the tRNAs, mainly owing to codon– anticodon interactions. This conclusion is supported by the observation that the main physical contacts between the mRNA and the ribosome during elongation are mediated by the codon–anticodon interactions59. This highlights the importance of codon–anticodon inter actions not only during decoding at the A­site but also at the P­site31,60,61 and the E­site22,32,62.

Activation‑energy barrier between PRE‑ and POST‑states. The PRE­states are separated from the POST­state by a high activation­energy barrier of 90 kJ mol–1 (REF. 63). EF­G reduces this barrier by establishing the TIPOST state

and accelerates the translocation rate by 104­ to 106­fold compared with spontaneous translocation (reviewed in REF. 64). Structures that possibly have a role in estab­ lishing the energy barrier are the bridges that connect the 30S and 50S subunits at the intersubunit face and the ribosomal proteins S12 and S13 (REF.  65), which are located close to the A­site and P­site tRNAs. However, studies have shown that disruption of some of the bridges66 or removal of S12 and S13 (REF. 65) only con­ fer a modest increase in the rates of both spontaneous translocation and back­translocation, which indicates that they have only a marginal role in establishing the energy barrier.

By contrast, it has been proposed that a structural element of the 16S rRNA might have a decisive role in creating the activation­energy barrier. A ridge of four bases, G1338­A­N­U1341 (where N represents any base), in the 30S head and the nucleotide A790 of the 30S platform form a gate that blocks movement of the tRNA anticodon stem between the P­ and E­sites67 (FIG. 5a,b). Four of the five nucleotides of this gate, which is referred to as the A790 gate, are universally conserved in all three domains of life. The A790 gate is 13.8 Å in width in the absence of EF­G (closed gate), which is too narrow to allow the passage of an RNA duplex, such as the anti­ codon stem of the P­site tRNA (which has a diameter of 20 Å). Therefore, this gate needs to open in order to enable movement of a P­site tRNA to the E­site. A series of published functional complexes in the absence and presence of EF­G have been analysed, which suggest that the A790 gate is closed in the absence of EF­G and in the POST­state31,46, but that it opens to a width of approxi­ mately 24 Å exclusively in the intermediate TIPOST state35. These findings are in clear agreement with a recent crys­ tal structure of translocation intermediates of bacterial ribosomes68 as well as with a first cryo­EM structure of a TIPOST ribosome containing two tRNAs116. Opening of the gate is accompanied and probably caused by the 18 ° swivel of the 30S head68, as the gate is closed in the non­ swivelled PRE­states (FIG. 5b). Swivelling of the 30S head not only opens the A790 gate, but also induces move­ ment of the tRNA2–mRNA complex on the 30S subunit from the A­ and P­sites to the P­ and E­sites, respectively, as recently shown by ensemble stopped­flow FRET57. X­ray structures of EF­G–70S complexes have shown that EF­G remains on the ribosome until the POST­state is reached31,32. In the POST­state, the A790 gate is closed (the width of the opening decreases to approximately 15 Å), which indicates that the energy barrier is re­estab­ lished before EF­G leaves the ribosome, thus preventing back­translocation of the tRNA2–mRNA complex to a PRE­state. Opening of the A790 gate in the TIPOST transi­ tion state is currently the most attractive explanation for how EF­G accelerates the translocation reaction, and the observations that are described here add a key structural correlate to this hypothesis.

A recent study suggests that transport of the tRNA2–mRNA complex through the A790 gate is facili­ tated by two universally conserved residues of the 16S rRNA, C1397 and A1503, which intercalate with mRNA bases only in the TIPOST transition state. A1503 inserts

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Nature Reviews | Microbiology

Swivelling 18°

A790

A1338- U1341

7°

Rotated

EF-G

EF-G

POST-states

Non-rotated

L1 stalk Head

4°

P/P A/A P/E A/P pe/E ap/P E/E P/P

Platform

Head

ASL 13.8 Å P/P P/E P/Ppe/E15.5 Å 23.6 Å

E/E 14.7 Å

L1 stalk

Open

PRE-states POSTClassical

a

b

c

H1 TI POST

Closed Intermediate Intermediate

Figure 5 | Ribosomal conformational changes during translocation. a | After peptidyl ‑transfer, the tRNAs are in the classical state (A/A and P/P), which establishes an equilibrium with the hybrid states H1 and H2 (H2 not shown) owing to intersubunit rotation. When elongation factor G (EF‑G) binds to one of these three PRE‑states, swivelling of the 30S head is induced, leading to the formation of the translocation intermediate TIPOST, which later resolves into the post‑translocational state (POST‑state) after a reversal of the head swivel and 30S back‑rotation. Top row, view of the 70S ribosome from the 30S solvent side showing the intersubunit movements. Bottom row, view from above the 70S ribosome showing the tRNA positions. b | Positions of the 16S rRNA base A790, which forms an important component of the A790 gate, corresponding to the ribosomal states that are shown in part a. The A790 gate is wide enough (23.6 Å) only

in the TIPOST intermediate state to allow passage of the anticodon stem of the tRNA from the P‑ to the E‑site on the 30S subunit during translocation. c | Positions of the L1 stalk in the open conformation (corresponding to the classical state of the tRNAs), closed conformation (corresponding to the hybrid states H1 and H2) and intermediate conformation (TIPOST and POST); the pivot point for rotation of the L1 stalk is indicated by the red dot. The following Protein Data Bank accessions were used for parts b and c: PRE classical (column 1), 3J0T and 3J0U46; PRE H1 (column 2), 3J10, 3J14 (REF. 46) and 3J0L114; TIPOST (column 3), 2XUX and 2XUY35; POST (column 4), 2WRI and 2WRJ31. ASL, anticodon stem‑loop; pe/E, pe indicates that the codon‑anticodon duplex takes a position between the P and E sites35; ap/P, indicates a position between the A‑ and P‑sites

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Exocyclic group A chemical group attached to a cyclic structure. For example, adenine contains an exocyclic amino group at position 6, and guanine contains a hydroxyl group at the same position.

between the second and third nucleotide of the E­site codon and C1397 between the +9 and +10 nucleotides68 (assuming the first nucleotide of the P­site codon is +1). Both of these 16S rRNA residues might be important for translocation by preventing back­sliding, thus function­ ing as ‘pawls’ as long as the gate is open (Supplementary information S4 (figure)), thereby cooperating with the ‘doorstop’ effect of EF­G.

Role of the L1 stalk. The L1 stalk undergoes dynamic structural transitions during the various stages of trans­ location. It can swing by approximately 30 ° around a pivot point of the stalk (located at the base of helix 76; (H76)), whereas the tip of the stalk can move by about 50 Å towards the intersubunit space. Three different L1 positions are observed31,35,37,46,69,70 (FIG. 5c): it adopts an open position during decoding and in the classical PRE­state; a closed position in the hybrid PRE­states (H1 and H2); and an intermediate position in the TIPOST and POST­state. Thus, the L1 stalk is proposed to function as a gate for the deacylated E­site tRNA, blocking release of the tRNA when it is in the closed position, but enabling free dissociation when it is in the

open position71. This hypothesis is consistent with the allosteric three­site model for the elongation cycle72, which posits that the E­site tRNA is only released when the A­site becomes occupied with the next aa­tRNA21–23,73, coinciding with opening of the L1 stalk during decoding. The coupling of different transloca­ tional states to distinct positions of the L1 stalk is clearly visible in X­ray and cryo­EM structures46, whereas FRET measurements have indicated that, at least under the in vitro conditions that were used, anticlockwise subunit rotation and L1 closure are only loosely coupled74,75.

As the L1 stalk is in contact with the deacylated tRNA in the H1, TIPOST and the POST­states (FIG. 5c), it has been suggested that it might carry the tRNA from the P­site to the E­site during translocation37,69. However, L1 is not an essential protein and its removal only leads to a 50% reduction in the growth rate of E. coli, which corresponds to a 50% reduction in poly(Phe) synthesis in vitro76. Furthermore, deletion of the L1 gene has no effect on EF­G­dependent translocation77, which sug­ gests that the L1 protein is unlikely to have an active role in tRNA transport from the P­site to the E­site. However, the importance of the L1 rRNA­binding site, which also makes contact with the tRNA, is unknown.

GTP hydrolysis. GTP hydrolysis on EF­G and EF4 is mediated by domains that are shared by both factors (FIG. 3c) and therefore probably follows identical path­ ways. GTP cleavage is not essential for tRNA movement, although EF­G­mediated translocation occurs at least fourfold faster with GTP compared with GDPNP78–80. How this acceleration is achieved is unclear, but it is modest, considering that EF­G­dependent transloca­ tion (with or without GTP hydrolysis) is at least four orders of magnitude faster than spontaneous transloca­ tion64 (BOX 1). GTP hydrolysis is primarily thought to be important for fast and efficient release of EF­G, which is required to enable the incoming ternary complex to bind to the ribosome. Although EF­G dependent GTP cleavage can precede translocation78, GTP hydrolysis and Pi release are not strictly coupled to the movement of the tRNA2–mRNA complex81.

Residues in the SRL of the 50S sub unit are impor­ tant for factor binding and are involved in trig­ gering GTP cleavage36,38,39,82,83. The SRL comprises the 2660 loop of H95 of the 23S rRNA, which contains the longest universally conserved stretch of 12 RNA nucleo­ tides82,84. Ribosomes in which the SRL is cleaved by the RNase toxin α­sarcin, as well as studies of SRL mutants, have revealed that the SRL is important for EF­Tu binding and essential for anchoring EF­G to the ribosome during the various conformational changes of the translocation process82,85,86. It has been shown that the exocyclic group of A2660, rather than the actual chemistry of this base, is crucial for GTP hydrolysis87, although the effects are indirect, as A2660 points away from the GTPase centre.

Our current understanding for the mechanism that triggers GTPase activity involves the hydrophobic resi­ dues Ile19 and Ile61 (E. coli nomenclature) of EF­G. These two amino acids are proposed to form a hydrophobic gate, which needs to open to enable His92 to approach GTP.

Box 1 | Spontaneous translocation and back‑translocation in vitro

Spontaneous translocation has been observed by several groups101,102, but it occurs at a rate that is at least four orders of magnitude slower than translocation catalysed by elongation factor G (EF‑G)–GTP (reviewed in REF. 64). Thiol‑modifying reagents, such as p‑chloromercuribenzoate103, or the absence of the ribosomal proteins S12 and S13 from the small ribosomal subunit65 accelerate the rate of spontaneous translocation, but the rate is still orders of magnitude slower than translocation catalysed by EF‑G–GTP.

Addition of deacylated tRNAs cognate to the codon at the E‑site can induce back‑translocation of ribosomes from the post‑translocational state (POST‑state) to a pre‑translocational state (PRE‑state)104,105. However, direct binding of a deacylated tRNA to the E‑site does not occur in vivo because deacylated tRNAs are always complexed with components of the translational machinery, such as the ribosomes or tRNA synthetases106. This is true despite the large fraction (30%) of deacylated tRNAs that are observed in minimal media107; in rich media, the percentage might be substantially lower. Thus, there is almost no pool of free deacylated tRNA under non‑starvation conditions because most of the tRNAs that are not bound to ribosomes or synthetases are fully charged with amino acids106,108.

Interestingly, when EF‑G is removed from a population of ribosomes in the post‑translocational state (POST‑state), the ribosomes partially fall back into the pre‑ translocational state (PRE‑state)95,104. This suggests that the energetic levels of PRE‑ and POST‑states are very similar, and that, in some cases, the PRE‑state might be slightly thermodynamically favoured over the POST‑state. The rates of spontaneous forward and reverse translocation are similar (about 0.5 to 2 × 10–3 s–1), which suggests that even small energetic increments could shift the equilibrium to either side. Such shifts are observed with antibiotics, which was first noted with sparsomycin‑triggered translocation109. Other examples are streptomycin, neomycin, paromomycin and viomycin, which shift the ribosome from the POST‑state to a PRE‑state, whereas hygromycin favours the POST‑state95,104.

The induction of back translocation by the addition of deacylated tRNAs to the POST‑state has been analysed in a time‑resolved cryo‑electron microscopy study, and the observed structures have been used to describe the conformational changes that occur during canonical forward translocation110. However, the validity of these interpretations is questionable for two main reasons. First, the induced back translocation is more than four orders of magnitude slower than an enzymatic translocation. Second, the energetic barriers between the various identified states are low (the energy landscape is flat, in striking contrast to EF‑G‑dependent translocation, which has high‑energy barriers between PRE‑ and POST‑states70,111). Therefore, there might only be a partial overlap between the structural intermediates of enzymatic translocation and non‑enzymatic back translocation.

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His92 positions a water molecule to attack the γ­phosphate of GTP. Three recent studies show His92 in an identical orientation pointing to the γ­phosphate of the GTP ana­ logue, GDPCP38,39,88 (FIG. 6a,b), which provides compelling evidence that these structures represent an active state of the GTPase centre. The studies also suggest how inter­ actions between residues of P­loop and switch I and II of EF­G cooperate with the SRL to open the hydrophobic gate. This enables His92 to move towards the γ­phosphate of GDPCP (reaching a distance of ~3 Å), which is stabi­ lized by hydrogen bonding to A2662. As a similar His92 arrangement was observed in crystal structures of isolated EF­Tu–GTP89, it is thought that GTPase activation follows the same mechanism in EF­G and EF­Tu.

Because the ‘active’ orientation of His92 is only observed in three translocation intermediates38,39,88 and the essential residues of the GTPase centre are positioned so that they are ready to cleave GTP, the time of GTP cleavage can now be identified: it occurs just before, or during, the formation of TIPOST (REF. 35), before the A790 gate fully opens39. Interestingly, His92 occupies a dif­ ferent orientation in one of the recent structures of the transition intermediates68: it is located 9 Å away from the γ­phosphate and points away from the bound nucleo­ tide, which indicates an inactive GTPase centre (FIG. 6b), similar to two unrotated states with an inactive GTPase centre, the POST­state31 and the EF­Tu–70S complex70 after GTP cleavage. The observation of an open A790

gate in the translocation intermediate38,39,88 and an inac­ tive GTPase centre (which occurs in the POST­state31) suggests that this structure represents a late transition intermediate just before arriving at the POST­state.

EF4 and back‑translocation The data available on 70S–EF4 complexes and the mecha­ nism of EF4 dependent back­translocation are still insuf­ ficient to provide a detailed description of the structural transitions that occur during this reaction. For example, the molecular basis by which EF4 might open the A790 gate to facilitate a reversal of the E­site tRNA to the P­site is unknown. However, a model for EF4­mediated back­translocation has been proposed28. By examining EF4­mediated back­translocation of POST­state ribo­ somes, the tRNAs were observed in a PRE­state that was unique to back­translocation. In this state, a deacylated tRNA was found in the P/P site, whereas the peptidyl­tRNA had moved beyond the A/A site to a posi­ tion known as the A/L site (L for LepA, the original name of EF4 (REF. 28)). In this position, the elbow of the A­site tRNA is displaced by ~14 Å towards the L12 stalk (FIG. 3c). When EF4 is released, the peptidyl­tRNA is predicted to fall back into the A/A position, which might be an important step for the re­mobilization of a stalled ribosome. These data indicate that EF4­dependent back­translocation is not a simple reversal of translocation; this view is also supported by FRET analysis of back ­translocation25.

Nature Reviews | Microbiology

SRL

His92 Ile61

P-loop

a b

GTPase centre of EF-G

GDPCP

His18 Ile19

Asp20

Inactive His92

Active GTPase conformation

Inactive GTPase conformation

Active His92

Active His92

SW II

γ-Ph

A2662G2661

γ-Ph SW I

Figure 6 | Mechanism of GTP hydrolysis on EF-G. a | The active GTPase centre of EF‑G in complex with a translocation intermediate in the presence of the non‑cleavable GTP analogue GDPCP (5ʹ‑guanosyl‑methylene‑triphosphate). The functional motifs of EF‑G are shown, namely the P‑loop, switch I (SW I) and switch II (SW II), together with a portion of the ribosomal sarcin–ricin loop (SRL). Interactions of His18 and the ‘catalytic’ His92 (Escherichia coli nomenclature) with nucleotides of the SRL are shown as dashed lines. In the active GTPase state, the catalytic His92 is oriented towards the γ‑phosphate (γ‑Ph) of GDPCP (distance 3 Å). Note that His18 and His92 interact with the backbone of the SRL (phosphate‑OH groups of G2661 and A2662, respectively; Protein Data Bank (PDB) accessions 4BTC and 4BTD32). b | Left panel, His92 from three crystal structures of the translocation intermediate38,39,88 have been aligned according to the bound nucleotide; His92 occupies an almost identical position in all three structures, which corresponds to an active GTPase centre (PDB accessions 4BTC38, 4JUW39 and 4KIX88). Right panel, in one translocation intermediate (PDB accession 3SFS68), His92 points away from the γ‑phosphate, similarly to the His92 (orange) in the inactive GTPase centre of the POST‑state (PDB accession 2WRI31).

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Physiological relevance of back‑translocation. What is the physiological relevance of a factor that can reverse the canonical translocation reaction? The wide distribu­ tion and high conservation of EF4 in bacteria argue for an important function. However, deletion of the encod­ ing gene (lepA) in E. coli has no phenotype when cells are grown in either rich or poor medium90. A first hint of the importance of EF4 came from a report showing that lepA is one of ten genes that are essential for survival of Helicobacter pylori in the hostile acidic environment of the stomach muscosa, which has a pH of 4 (REF. 91). Low pH is equivalent to high H+ concentrations, suggesting that EF4 could have an important physiological role at high ionic strength, which could be caused by high intracellular levels of K+ and Mg2+. For example, under hyperosmotic conditions, the intracellular concentra­ tions of Mg2+ and K+ (together with glutamate) increase three­ to sevenfold92,93. A change in K+ concentration over a wide range has only a marginal effect on protein synthesis in vitro. By contrast, an increase in Mg2+ leads to the ribosome becoming more compact and less flex­ ible, resulting in an increase in error rate and a decrease in translation rate owing to both decelerated ribosome movement and an increase in the number of stalled ribosomes on mRNAs94,95.

A recent analysis showed that EF4 has no effect on the rate of elongation under physiological Mg2+ concentra­ tions (4.5 mM), whereas it accelerates protein synthesis by about fivefold when the Mg2+ concentration is increased threefold in vitro26. These data suggest that EF4 might function in recognizing ribosomes that are stalled either in the PRE­ or the POST­state, and that it re­mobilizes them, thus recycling both the mRNA and the associ­ ated ribosomes of the polysome. It was shown in vivo and in vitro that EF4 does not reduce misincorporation errors26,96, whereas a previous study 24 showed that EF4 increases the fraction of functional proteins produced in the cell (which could be due to a reduction in misincor­ poration rate). However, this effect was only observed at increased Mg2+ concentrations, but not in the presence of aminoglycosides, which are known to increase the misincorporation rate97. A possible explanation is that EF4 indirectly leads to increased synthesis of functional proteins by preventing the misfolding of proteins (rather than counteracting misincorporations). Consistent with this hypothesis, protein misfolding is known to occur when the ribosome is subject to unscheduled stalls98.

The relationship between increased Mg2+ concentra­ tion and EF4 activity is consistent with the pheno type that is associated with LepA­depleted (ΔlepA) E. coli mutants grown in competition with wild­type cells in media containing 100 mM Mg 2+ at pH 6. Wild­type cells show a strong growth advantage under these con­ ditions, whereas there was no substantial difference between wild­type and ΔlepA mutants in medium that contains 1 mM Mg2+ at pH 7 (REF. 26). Surprisingly, the intracellular concentration of EF4 in vivo is the same during growth under physiological and hyperosmotic conditions. However, during physiological growth con­ ditions, almost all EF4 proteins are associated with the membrane, whereas the majority of EF4 is found in

the cytoplasm under hyperosmotic conditions26. This sug­ gests that the membrane is a storage vessel for EF4 under optimal growth conditions and that EF4 is liberated when the Mg2+ concentration rises to unfavourable levels.

The lack of EF4 orthologues in archaea and the cyto­ plasm of eukaryotes might be related to the fact that hyperosmotic conditions generally leave the intra cellular concentrations of K+ and Mg 2+ largely unchanged99. However, the EF4 orthologue in mitochondria and chloro plasts might have the same function as EF4 in bacteria. Depending on the rates of respiration and photo synthesis, the inner membrane potential of these organelles can change sharply, which affects the pH of the cytosol close to the membrane where protein synthesis occurs. Similarly to E. coli EF4, the mitochondrial homo­ logue Guf1 is found at the inner membrane. A Δguf1 yeast strain has a reduced growth rate under suboptimal temperatures and starvation conditions. Protein synthe­ sis is only marginally perturbed in the knockout strain, but the production of functional proteins is reduced100. Similarly to bacterial EF4 (REF. 98), this would suggest that Guf1 might also reactivate stalled ribosomes and thereby enhance the production of functional proteins. The pro­ posed ability of EF4 to resolve stalled ribosomes when the pH and Mg2+ concentrations are unfavourable has two important consequences: it could accelerate protein syn­ thesis by mobilizing stalled ribosomes and it could also prevent co­translational misfolding. However, it should be noted that the evidence of a role for EF4 in rescuing stalled ribosomes is suggestive rather than direct, thus further studies are required to confirm this potential role.

Summary and outlook The opposing functions of EF­G and EF4, which trig­ ger translocation and back­translocation, respectively, are mediated by their specific domains (domain IV of EF­G and the CTD of EF4 (FIG. 3)). During trans­ location, EF­G reduces the activation­energy barrier between the PRE­ and POST­states, probably by open­ ing of the A790 gate during swivelling (FIG. 5B), which enables the tRNAs to translocate to the POST­state. Domain IV of EF­G enters the A­site as soon as the tRNAs have moved from the PRE­ to the POST­state and thereby blocks back translocation. The exact details of the mechanism of EF4­mediated back­transloca­ tion of the tRNA2–mRNA complex have not yet been resolved. Deacylated tRNA and peptidyl­tRNA in the E­ and P­sites are moved to the P­ and A­sites, respec­ tively, and it seems as though the CTD of EF4 halts the peptidyl­tRNA at the A­site and drags the elbow of the peptidyl­tRNA beyond the A­site to the A/L posi­ tion (FIG. 3c; Supplementary information S2 (figure)). The data suggest that EF4­triggered back translocation is not a simple reversal of translocation. However, we have much to learn about the structural transitions that occur during this reaction before the principles of back­translocation can be elucidated. Furthermore, evidence so far suggests that EF4 can bind to both PRE­ and POST­state ribosomes, but whether one or the other is the preferential target of EF4 remains an unanswered question27,28.

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Although we now know a great deal about the mechanistic details of translocation, there is one major step about which we still know very little, although it is crucial for a complete understanding of both translo­ cation and back­translocation: the early contacts of an elongation factor with the ribosome. A recent paper115 provides some answers for EF­G: a PRE­state containing EF­G and two tRNAs in the hybrid sites shows an ~12° rotation of the 30S subunit but a negligible swivelling of head by only 3°. These are features of a ribosome– EF­G complex before the formation of a translocational

intermediate. The tip of EF­G domain IV makes strong contacts with the anticodon loop of the A­site tRNA but has not yet entered the A­site. Despite these important insights, it is still unclear which ribosomal contacts are required to initiate the translocation process, includ­ ing 30S head swivelling, and to coordinate movement of both the tip of domain IV and the tRNA2–mRNA complex.

Solving this problem will represent major progress in our understanding of translocation, which will also have implications for back­translocation.

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Acknowledgements The authors thank J. Harms (Hamburg) N. Polacek (University Bern, Switzerland) and T. Sprink (Charité, Berlin) for help and discussions. H.Y. and C.M.T.S acknowledge the support of the Deutsche Forschergruppe (DFG), Forschergruppe 1805, and Y.Q. is grateful for research grants from the Major State Basic Research of China 973 project (grant 2012CB911000) and the National Natural Science Foundation of China (grants 31270847 and 31322015).

Competing interests statement The authors declare no competing interests.

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