Translocation and the Elongation Cycle
A ribosome making a single hemoglobin chain must read 141 codons in under a minute. It must do this without ever slipping a frame. Each cycle it must move exactly three nucleotides — never two, never four. At the same time, it carries two tRNAs the size of small proteins. This precision is not free. It is bought with GTP hydrolysis and a molecular machine called EF-G. EF-G physically ratchets the ribosome forward. Translocation is a step of the elongation cycle. It happens after the ribosome forms a new peptide bond. At this step, the ribosome moves the mRNA and its attached tRNAs. They shift exactly one codon in the 3′ direction. Without it, the ribosome would simply add amino acids to a static mRNA position. The genetic code would then be unreadable as a sequence. The event links two things that must never be decoupled. These are the mechanical advance of the mRNA reading frame. The other is the physical repositioning of tRNAs among three ribosomal sites — A, P, and E. This article dissects translocation mechanistically. It covers the elastic, spring-loaded intermediate states of the ribosome. It also covers the motor role of elongation factor EF-G (EF-2 in eukaryotes). It examines the biophysical evidence for this process. This evidence ranges from single-molecule FRET to cryo-EM snapshots. Together, this evidence converted a black-box "the ribosome moves" idea. It became a structurally resolved, multi-step ratchet mechanism. It builds directly on Peptide Bond Formation and Peptidyl Transferase and Elongation and Codon Recognition. It also sets up Termination and Release Factors and Polysomes and Translational Efficiency.
- Translocation moves mRNA and tRNAs by exactly one codon (3 nucleotides) per elongation cycle, driven by EF-G·GTP.
- The ribosome cycles through A, P, E tRNA sites; translocation shifts peptidyl-tRNA A→P and deacylated tRNA P→E.
- EF-G binding and GTP hydrolysis trigger a large-scale ratcheting/swiveling motion of the ribosomal subunits.
- Intermediate "hybrid states" (A/P, P/E) exist before full translocation, discovered by single-molecule FRET.
- Cryo-EM captured EF-G frozen mid-translocation, visually confirming the ratchet mechanism proposed from biochemistry.
- Fusidic acid and viomycin are antibiotics that trap distinct translocation intermediates, proving the stepwise model.
Translocation is a GTP-powered, two-part ratchet — spontaneous hybrid-state formation followed by EF-G-catalyzed locking — that advances the ribosome exactly one codon while shuttling tRNAs through the A, P, and E sites.
The core idea
The elongation cycle of translation has three repeating steps: aminoacyl-tRNA selection (decoding), peptide bond formation, and translocation. Translocation is the mechanical "reset" step. After peptidyl transfer, the ribosome holds a peptidyl-tRNA in the A site and a deacylated tRNA in the P site, both still base-paired to their codons on an unmoved mRNA. Translocation must accomplish three coupled movements simultaneously: the peptidyl-tRNA shifts from the A site to the P site, the deacylated tRNA shifts from the P site to the E (exit) site, and the mRNA advances by one codon so a fresh codon enters the now-empty A site. The key conceptual move — established over four decades of biochemistry, genetics, and structural biology — is that translocation is not a single concerted jump but a multi-step ratchet. The two ribosomal subunits (30S and 50S in bacteria) rotate relative to each other, the head domain of the small subunit swivels independently, and tRNAs pass through transient "hybrid" states (A/P and P/E) before landing in their final classical positions. EF-G (elongation factor G, called eEF2 in eukaryotes and archaea) is a GTPase that binds the ribosomal A site, and its GTP hydrolysis converts thermal, Brownian ratcheting motions that occur spontaneously into a directional, essentially irreversible forward step. The result is a cycle with remarkable fidelity: translocation must move the mRNA by precisely three nucleotides, coupling this to the codon-anticodon geometry so that reading frame is preserved for hundreds to thousands of cycles per mRNA. Any slippage — extra or missing nucleotides moved — causes a frameshift, scrambling every downstream codon.
Mechanism
Step 0 — Starting state (post-peptidyl transfer). Following peptide bond formation (see Peptide Bond Formation and Peptidyl Transferase), the ribosome is in the "classical" pre-translocation (PRE) state: peptidyl-tRNA occupies the A site, deacylated tRNA occupies the P site, both tRNA anticodons paired to their mRNA codons, and the E site is empty. The peptide has just been transferred from the P-site tRNA onto the A-site aminoacyl group, so the amino acid chain now hangs off the A-site tRNA.
Step 1 — Spontaneous ratcheting and hybrid-state formation. Even before EF-G arrives, the ribosome undergoes thermally driven, reversible fluctuations. The 30S subunit rotates by roughly 6-9° relative to the 50S subunit (the "ratchet" motion, first described by Frank and Agrawal from cryo-EM), and within the 30S the "head" domain swivels independently. These motions allow the acceptor ends of the tRNAs (the CCA-amino-acid end, buried in the 50S subunit) to move into the P and E sites while the anticodon ends (still in the 30S subunit) remain paired to their codons. This produces the hybrid states:
- A/P hybrid state: acceptor end in 50S P site, anticodon end still in 30S A site.
- P/E hybrid state: acceptor end in 50S E site, anticodon end still in 30S P site.
This spontaneous, EF-G-independent step was the single biggest surprise of modern translocation research — it means the ribosome partially "pre-translocates" itself using ordinary thermal motion, and EF-G's job is to capture and complete this motion rather than to initiate it from scratch.
Step 2 — EF-G·GTP binding. EF-G, a multidomain GTPase structurally similar in shape to a tRNA bound to elongation factor EF-Tu (a striking case of molecular mimicry), binds in the A site of the ribosome, which is now vacated of its anticodon-stem-loop position by the hybrid-state shift. Domain IV of EF-G inserts deep into the decoding center near the mRNA and the A-site codon.
Step 3 — GTP hydrolysis and conformational trapping. GTP hydrolysis on EF-G, stimulated by the sarcin-ricin loop of the 23S rRNA, triggers a conformational change in EF-G. This locks in and drives to completion the swiveling of the 30S head domain, translocating the anticodon ends of the tRNAs (and the mRNA still base-paired to them) from the A and P sites into the P and E sites. Domain IV of EF-G acts as a wedge/pawl that prevents the tRNA-mRNA complex from sliding backward — a ratchet pawl in the literal biophysical sense.
Step 4 — Pi release and EF-G ejection. Inorganic phosphate is released, EF-G's affinity for the ribosome drops, and EF-G·GDP dissociates. The ribosome relaxes back from the rotated to the non-rotated (classical) conformation, locking tRNAs into their final classical P and E sites, with the new codon exposed in the A site.
Step 5 — E-site tRNA release. The deacylated tRNA, now in the classical E site, is released from the ribosome — a step influenced by whether a new aminoacyl-tRNA has entered the A site (allosteric three-site model; there is ongoing discussion about exactly when E-site release occurs relative to A-site occupation).
Net result: the mRNA has advanced 3 nucleotides, peptidyl-tRNA sits in the P site ready for the next peptide bond, and the A site is open for the next aminoacyl-tRNA-EF-Tu-GTP ternary complex, restarting the cycle described in Elongation and Codon Recognition. Energetically, one GTP is hydrolyzed per translocation event (in addition to the GTP hydrolyzed by EF-Tu during decoding), making the total GTP cost of adding one amino acid to a growing chain two GTP equivalents (plus the ATP-consuming aminoacylation of tRNA upstream). Translocation itself proceeds at roughly ~5-20 elongation cycles per second in bacteria at 37°C, meaning each translocation step completes in tens of milliseconds.
The evidence
1. Puromycin and the two-site model (Watson, Rich, and colleagues, 1960s). Before translocation was understood mechanically, the two-site (A/P) model of the ribosome was tested using puromycin, an antibiotic that mimics the 3′ end of aminoacyl-tRNA and accepts the peptide from peptidyl-tRNA. Puromycin reacts efficiently with peptidyl-tRNA in the P site, but only after EF-G action does the previously A-site peptidyl-tRNA become puromycin-reactive as a P-site occupant — behavior consistent with a physical A-to-P movement rather than a static labeling model. This provided the first functional (not structural) evidence for a discrete, EF-G-dependent movement step.
2. GTPase and antibiotic dissection (1970s-1980s). Biochemists showed that EF-G-dependent GTP hydrolysis is tightly coupled to translocation: non-hydrolyzable GTP analogs (like GDPNP) allow EF-G binding but block translocation, showing hydrolysis — not just binding — drives the forward step. The antibiotic fusidic acid locks EF-G onto the ribosome after GTP hydrolysis and Pi release, trapping a single defined post-translocation-like EF-G·GDP·ribosome complex; this became a key tool to isolate and structurally characterize the EF-G-bound intermediate. Viomycin, in contrast, traps the ribosome in a hybrid-state-like conformation, showing that the rotated/hybrid intermediate is a distinct, stabilizable state, not just a computational abstraction.
3. Cryo-EM snapshots of ratcheted ribosomes (Frank and Agrawal, 2000). Using cryo-electron microscopy of ribosomes stalled with EF-G and GTP analogs, Joachim Frank and Rajendra Agrawal directly visualized a rotated conformation of the 30S subunit relative to the 50S subunit — the physical "ratchet" — providing the first structural picture that a large-scale subunit rotation accompanies translocation. This was a landmark because it converted an abstract kinetic step into a visible molecular motion.
4. Single-molecule FRET reveals hybrid states (Puglisi lab and Noller lab, mid-2000s). By labeling tRNAs with fluorescent donor/acceptor dyes and the ribosome with immobilized surface tethers, researchers used single-molecule fluorescence resonance energy transfer (smFRET) to watch individual ribosomes in real time. They observed that, even without EF-G, ribosomes spontaneously and reversibly fluctuate between the classical (A/A, P/P) state and hybrid (A/P, P/E) states — confirming that hybrid-state formation is a thermally driven equilibrium that precedes and is captured by EF-G, rather than something EF-G forces from scratch. This was the direct kinetic proof of the two-step (spontaneous fluctuation + EF-G-driven locking) ratchet mechanism.
5. High-resolution cryo-EM and X-ray structures of EF-G-bound ribosomes (2009-2015, multiple groups including Ramakrishnan, Cate, Frank). Structures resolved to near-atomic detail showed EF-G domain IV inserted into the decoding center exactly where the anticodon stem-loop of the A-site tRNA had been, physically preventing back-slippage — direct structural confirmation of the "pawl" function proposed from kinetic data, and revealing that EF-G's shape mimics an aminoacyl-tRNA-EF-Tu ternary complex (molecular mimicry, first proposed on structural grounds by comparing EF-G and EF-Tu-tRNA crystal structures in the 1990s). Together, these lines of evidence — functional (puromycin reactivity), pharmacological (antibiotic trapping), structural (cryo-EM ratchet), and single-molecule kinetic (smFRET hybrid states) — independently converge on the same mechanistic model, which is the gold standard for establishing a molecular mechanism in modern biology.
Worked examples
Example 1 — Counting GTP cost and elongation time for a real protein. Consider the beta-globin polypeptide, 146 amino acids long (note: mature hemoglobin beta chain is 146 residues, not the commonly misquoted 141). Each amino acid addition after initiation requires:
- 1 GTP hydrolyzed by EF-Tu during aminoacyl-tRNA delivery and codon proofreading
- 1 GTP hydrolyzed by EF-G during translocation
So for 145 elongation cycles (146 residues added, but the initiator methionine does not require a translocation/EF-Tu cycle the same way — count elongation cycles as chain length minus 1): ``` Elongation cycles = 146 - 1 = 145 GTP per cycle = 2 (EF-Tu + EF-G) Total elongation GTP = 145 × 2 = 290 GTP ``` At a bacterial elongation rate of ~15 amino acids/second (a typical E. coli value at 37°C), synthesizing the full 146-residue chain takes: ``` Time = 145 cycles ÷ 15 residues/sec ≈ 9.7 seconds ``` This calculation shows why translation is so GTP-expensive relative to DNA replication (which uses no analogous per-monomer GTP-driven translocase step) — the ribosome pays continuously for both fidelity (EF-Tu proofreading) and mechanical advancement (EF-G translocation), a cost thought to buy the low error rate of roughly 1 mistranslated codon per 10,000, itself dependent on proper coupling between decoding and translocation.
Example 2 — Diagnosing a frameshift mutant using translocation logic. Suppose a bacterial gene encoding a 200-residue enzyme is mutagenized, and a mutant strain produces a truncated, nonfunctional 43-residue peptide with an entirely scrambled C-terminal sequence compared to wild type, ending in a premature stop. Two hypotheses: (a) a nonsense (point) mutation created a premature stop codon in-frame; (b) an insertion or deletion mutation shifted the reading frame, and translocation is now reading the "wrong" three-nucleotide windows. Reasoning through the translocation mechanism distinguishes these:
- If (a) nonsense mutation: the first 42 residues would be identical to wild type, since translocation still moves 3 nucleotides at a time along an unperturbed frame up until the new stop codon.
- If (b) frameshift: the reading frame downstream of the insertion/deletion is entirely different, because translocation faithfully moves exactly 3 nt per cycle regardless of the frame — it cannot "know" the original frame was lost. All codons after the lesion are misread, generating a scrambled sequence, and a stop codon is encountered in the new frame purely by chance (roughly every 20-someodd codons on average, since 3 of 64 codons are stops).
Because the mutant peptide is scrambled at the C-terminus rather than truncated with an otherwise-normal N-terminal-matching sequence, the observation is diagnostic of a frameshift, not a nonsense mutation — precisely because translocation is a fixed-step, sequence-blind ratchet that cannot self-correct a shifted frame. This logic (first used by Crick, Barnett, Brenner, and Watts-Tobin in 1961 in the rII frameshift experiments with acridine mutagens in bacteriophage T4) was itself part of how the triplet nature of the code — the "read 3 nucleotides per step" rule underlying translocation — was established.
Common misconceptions & traps
| Misconception | Why it's wrong |
|---|---|
| "Translocation is a single instantaneous jump." | It is a multi-step process: spontaneous hybrid-state formation, EF-G binding, GTP hydrolysis, subunit ratcheting, Pi release, and relaxation — each kinetically distinct and independently measurable. |
| "EF-G forces the ribosome to move; without it nothing happens." | Hybrid states form spontaneously (thermally) even without EF-G, as shown by smFRET; EF-G's role is to trap and complete this pre-existing fluctuation directionally, using GTP hydrolysis to make it essentially irreversible. |
| "GTP hydrolysis directly powers the mechanical movement, like ATP powering myosin." | GTP hydrolysis on EF-G mainly triggers a conformational switch and biases/traps a Brownian ratchet toward the forward direction; the mRNA-tRNA movement itself is largely powered by thermal fluctuation, not by GTP hydrolysis acting as a lever directly. |
| "The mRNA moves independently of the tRNAs during translocation." | The mRNA is dragged along because it remains base-paired to the tRNA anticodons throughout the anticodon-end movement; tRNA and mRNA movement are mechanically coupled, not separate events. |
| "Translocation always moves exactly one tRNA-sized step and nothing can go wrong." | Ribosomes do occasionally undergo programmed or error-induced frameshifting (-1 or +1), some directed by mRNA slippery sequences and pseudoknots — translocation fidelity is high but not absolute. |
The scientist behind it
Origins · 1 minJoachim Frank and how this was discovered
Joachim Frank pioneered single-particle cryo-electron microscopy image-reconstruction methods in the 1970s-1980s, developing the computational algorithms that let thousands of individual, randomly oriented frozen ribosome particles be aligned and averaged into a three-dimensional structure. Working with Rajendra Agrawal and colleagues at the Wadsworth Center in Albany, New York, Frank applied these methods in the late 1990s and 2000 to trap ribosomes at defined points in the elongation cycle — including EF-G-bound, ratcheted states — producing the first direct visual evidence of the intersubunit rotation that underlies translocation. He shared the 2017 Nobel Prize in Chemistry (with Jacques Dubochet and Richard Henderson) for developing cryo-EM as a general high-resolution structural technique, a method that subsequently transformed structural biology far beyond the ribosome.
Where this matters today
Antibiotic development. Because bacterial EF-G differs enough from eukaryotic eEF2 to allow selective drug targeting, translocation is a validated antibiotic target. Fusidic acid, used clinically (particularly for staphylococcal infections including some MRSA strains), works by trapping EF-G on the ribosome after GTP hydrolysis, freezing translocation and halting bacterial growth. Understanding the precise conformational cycle allows rational design of next-generation translocation inhibitors as resistance to older drugs spreads.
Diphtheria toxin and ADP-ribosylation. Diphtheria toxin and Pseudomonas exotoxin A inactivate eukaryotic eEF2 (the human counterpart of EF-G) by ADP-ribosylating a unique modified histidine residue called diphthamide. This blocks translocation in host cells entirely, halting all protein synthesis — the molecular basis of diphtheria's lethality, and a direct clinical illustration of why understanding the translocation mechanism matters for toxinology and vaccine design (the diphtheria toxoid vaccine).
mRNA vaccine and therapeutic design. Efficient translocation kinetics affect how fast and how accurately ribosomes translate a synthetic mRNA (such as those used in COVID-19 mRNA vaccines). Codon-optimization strategies for therapeutic mRNAs consider translocation and decoding kinetics together, since suboptimal codons can cause ribosome stalling, reduce protein yield, and even trigger ribosome quality-control pathways.
Understanding programmed ribosomal frameshifting. Many viruses, including HIV and coronaviruses, use mRNA secondary structures ("slippery sequences" and pseudoknots) to force the ribosome to translocate by −1 nucleotide at defined sites, producing frameshifted fusion proteins essential for viral replication. Because researchers understand the normal three-nucleotide translocation ratchet in mechanistic detail, they can identify exactly how these viral elements subvert it — and this has become an active target for antiviral drug design.
Foreshadowing termination and polysomes. The same translocation cycle that repeats through elongation eventually delivers a stop codon into the A site, where no aminoacyl-tRNA exists to decode it; this triggers the entirely different machinery of Termination and Release Factors. Meanwhile, because each ribosome physically clears the initiation site as it translocates forward, multiple ribosomes can simultaneously translate a single mRNA in tandem, forming the polysomes central to Polysomes and Translational Efficiency and to how cells scale up protein output from a single message.
Ask the tutor: How exactly does EF-G's domain IV prevent the ribosome from slipping backward during translocation, at the level of atomic contacts?
Why does translocation move exactly 3 nucleotides and not some other number?
The genetic code is read in nonoverlapping triplets (codons), a fact established by Crick, Barnett, Brenner, and Watts-Tobin's 1961 frameshift experiments with acridine dyes in bacteriophage T4. Since each tRNA anticodon pairs with exactly three mRNA bases, and translocation moves the anticodon-mRNA duplex as a coupled unit from the A/P sites into the P/E sites, the physical geometry of codon-anticodon pairing fixes the step size at three nucleotides. EF-G's domain IV and the ribosome's decoding center are structurally built around this triplet geometry, so the "3-nucleotide rule" is enforced by the molecular architecture of the translocation machinery itself, not by a separate counting mechanism.
Is translocation identical in bacteria and in humans (eukaryotes)?
The core mechanism — GTPase-driven ratcheting through hybrid states, A-P-E site tRNA movement, and one-codon mRNA advancement — is conserved from bacteria to humans, reflecting a deeply ancient, universal ribosomal mechanism. The eukaryotic and archaeal GTPase is called eEF2 rather than EF-G, and it carries a unique modified histidine residue (diphthamide) that bacterial EF-G lacks. This difference is medically important: diphtheria and Pseudomonas exotoxins specifically ADP-ribosylate diphthamide, selectively shutting down eukaryotic (host) translocation while leaving bacterial EF-G untouched, and conversely several antibiotics (like fusidic acid) target bacterial EF-G with little effect on human eEF2.
What would happen if translocation occurred without EF-G at all?
In experiments using non-hydrolyzable GTP analogs or EF-G-depleted extracts, ribosomes can still show limited, reversible rotation and hybrid-state formation, driven purely by thermal motion, but they cannot complete a full, committed translocation step. Without functional EF-G, the ribosome effectively stalls after peptide bond formation: mRNA does not advance, the A site is not cleared, and no further aminoacyl-tRNA can be accommodated. This is precisely why EF-G-targeting antibiotics like fusidic acid are bactericidal or bacteriostatic — they trap ribosomes in a stalled, post-hydrolysis EF-G-bound state, halting bacterial protein synthesis and growth.
How was it proven that hybrid states are real intermediates and not just an artifact of cryo-EM sample freezing?
The strongest evidence came from single-molecule FRET performed on freely diffusing or surface-tethered ribosomes in solution, at physiological or near-physiological temperature, without any chemical fixation. Researchers observed real-time, reversible fluorescence signal changes consistent with spontaneous transitions between classical and hybrid tRNA configurations, occurring on a timescale of milliseconds to seconds, entirely independent of the cryo-EM freezing process. Because this kinetic behavior was seen in solution using an entirely different physical technique (fluorescence rather than electron microscopy), and agreed quantitatively with the structural snapshots, the hybrid state was validated as a genuine solution-phase intermediate rather than a freezing artifact.
Does translocation ever go in reverse, and does this matter biologically?
Yes — because hybrid-state formation is a thermally reversible equilibrium before EF-G locks it in, ribosomes can occasionally slip backward as well as forward, and even after EF-G has driven translocation forward, rare back-translocation events have been documented, particularly under certain antibiotic or ribosome-mutant conditions. Under normal cellular conditions this reverse slippage is rare and functionally negligible because EF-G's GTP-hydrolysis-driven trapping step makes forward translocation strongly favored (essentially irreversible in the cell). However, engineered or antibiotic-perturbed ribosomes that allow more back-translocation are valuable research tools for dissecting the ratchet mechanism's individual kinetic steps.
How does translocation relate to translational fidelity and error rates?
Translocation fidelity is distinct from but linked to decoding fidelity. Decoding fidelity (during aminoacyl-tRNA selection, governed by EF-Tu) determines whether the correct amino acid is added; translocation fidelity determines whether the reading frame is correctly maintained. Typical bacterial missense (wrong-amino-acid) error rates are around 1 in 1,000 to 1 in 10,000 codons, while frameshifting errors during translocation are considerably rarer under normal conditions, though certain mRNA sequences (slippery sequences, rare codons, or specific antibiotics like streptomycin) can elevate frameshift rates substantially, sometimes exploited naturally by viruses for programmed ribosomal frameshifting.
Why is EF-G structurally similar in shape to a tRNA bound to EF-Tu?
X-ray crystal structures in the 1990s revealed that EF-G's domain III-V region mimics the overall shape and dimensions of an aminoacyl-tRNA molecule bound to EF-Tu·GTP, despite having no sequence similarity to tRNA. This is a striking example of molecular mimicry: because EF-G occupies the same physical space in the ribosome (spanning the A site and the factor-binding region) that an incoming aminoacyl-tRNA-EF-Tu complex would occupy, evolution appears to have shaped EF-G to fit the same structural "socket" on the ribosome, allowing it to insert domain IV deep into the decoding center exactly where the tRNA anticodon stem-loop normally sits.