At a stop codon in the mRNA (UAA, UAG, UGA), there is no matching tRNA anticodon. Instead, protein release factors (RFs) decode the stop and catalyze hydrolysis of the peptidyl-tRNA bond in the P site—releasing the finished polypeptide. Then the ribosome is recycled for another round.
Small subunit (decoding) Large subunit (catalysis)
A P E sites Peptidyl Transferase Center (PTC)
mRNA → → → [ ] [ ] [ ] [ catalytic rRNA + factors ]
^ ^
| +-- peptidyl-tRNA
+------- stop codon enters A site
Step-by-step: Bacteria (70S; 30S+50S)
Cast:
- RF1 recognizes UAA/UAG (class-1 RF)
- RF2 recognizes UAA/UGA (class-1 RF)
- RF3-GTP helps dissociate RF1/RF2 after release (class-2 RF, GTPase)
- RRF (ribosome recycling factor) + EF-G-GTP and IF3 recycle subunits
1) A-site stop codon arrives.
The mRNA has advanced so that UAA/UAG/UGA sits in the A site; the P site holds the peptidyl-tRNA; E site holds deacylated tRNA or is empty.
2) tRNA cannot bind—RF binds instead.
- If UAA/UAG → RF1 enters A site.
- If UAA/UGA → RF2 enters A site.
Each RF presents a conserved GGQ motif on a flexible loop aimed toward the large-subunit PTC.
3) Decoding center “reads” the stop.
The 30S decoding center (rRNA bases A1492, A1493, G530 in 16S rRNA) rearranges to stabilize the RF–codon interaction.
- RF1 carries a PxT motif, RF2 an SPF motif—these contact the stop triplet in the A site and exclude near-cognate tRNAs.
4) Accommodation and catalysis setup.
Protein/nucleotide rearrangements swing the RF’s GGQ loop into the peptidyl transferase center (PTC) of 23S rRNA, close to the ester bond linking the nascent chain to the P-site tRNA’s 3′-A76.
5) Peptide release (the chemistry).
The GGQ motif and ribosomal rRNA position a water molecule as the nucleophile.
- The water attacks the carbonyl of the ester linkage → hydrolysis → polypeptide released from P-site tRNA.
6) RF3-GTP promotes factor turnover.
- RF3-GTP binds, triggering conformational changes that eject RF1/RF2.
- RF3 hydrolyzes GTP and dissociates.
7) Ribosome recycling.
- RRF binds; EF-G-GTP drives a large-scale rearrangement that splits 50S off the 30S.
- IF3 binds 30S, ejects deacylated tRNA and mRNA, resetting for initiation.
Step-by-step: Eukaryotes (80S; 40S+60S)
Cast:
- eRF1 recognizes all three stops (class-1 RF)
- eRF3-GTP is the GTPase escort that delivers eRF1 and times catalysis
- ABCE1 (Rli1) ATPase recycles the ribosome
1) A-site stop codon arrives.
mRNA places UAA/UAG/UGA in the 40S A site.
2) eRF1·eRF3-GTP complex binds the A site.
- eRF1 mimics tRNA geometry and occupies the A site.
- eRF3-GTP chaperones eRF1 and gates timing.
3) Decoding center recognition.
The 40S decoding center (18S rRNA) stabilizes eRF1 on the stop triplet via conserved motifs in eRF1 (NIKS for the stop triplet; YxCxxxF contributes to specificity). This excludes near-cognate tRNAs.
4) Accommodation and catalysis setup.
Conformational changes allow the GGQ loop of eRF1 to enter the 60S PTC, aligned with the P-site peptidyl-tRNA ester.
5) Peptide release.
As in bacteria, the GGQ positions water for ester-bond hydrolysis → nascent polypeptide is released.
6) GTP hydrolysis and factor departure.
eRF3 hydrolyzes GTP, helping dissociate from the ribosome; eRF1 leaves after catalysis.
7) Ribosome recycling.
The ABCE1 ATPase binds the post-termination complex and splits 80S into 40S + 60S; initiation factors clear mRNA/tRNA and prepare for a new round.
Special cases (how “stop” is overridden)
- Selenocysteine (Sec, U) at UGA: a SECIS RNA element plus dedicated factors (SelB/eEFSec and tRNA^Sec) deliver Sec-tRNA to the A site, out-competing eRFs, so translation continues (not terminate).
- Pyrrolysine (Pyl, O) at UAG in certain archaea/bacteria: a dedicated tRNA^Pyl and PylRS supply Pyl-tRNA to the A site, preventing RF binding → insertion instead of stop.
- Programmed readthrough: Specific RNA signals (downstream pseudoknots, heptanucleotide contexts, or near-cognate tRNAs) can promote suppression of stop and continue translation (used by some viruses and a few cellular genes).
- No-stop / stalled rescue:
- Bacteria: tmRNA–SmpB (trans-translation) or ArfA/ArfB systems rescue ribosomes on nonstop mRNAs.
- Eukaryotes: Nonstop decay (Ski7/exosome) and No-go decay (Pelota/Dom34–Hbs1) clear stalled ribosomes; ABCE1 helps split and recycle.
The chemistry at the heart (why hydrolysis works)
- The ester bond between the peptide C-terminus and the 3′-OH of A76 of P-site tRNA is positioned in the PTC.
- GGQ (Gly-Gly-Gln) from the release factor plus rRNA nucleotides align a water molecule for a base-catalyzed attack on the carbonyl carbon → tetrahedral intermediate → collapse → products: free peptide (with new C-terminal –COO⁻/–COOH) and deacylated tRNA.
One-screen mental model
Decode (small subunit) → Decide (stop? RF binds) → Hydrolyze (GGQ + water at PTC) → Release (polypeptide) → Reset (RF turnover + subunit splitting)
- Bacteria: RF1/2 → RF3 → RRF+EF-G → IF3
- Eukaryotes: eRF1+eRF3 → ABCE1