Biochemistry chapters
Protein synthesis (translation)
How the genetic code in mRNA is read into protein: features of the code, tRNA and aminoacyl-tRNA synthetases, prokaryotic and eukaryotic ribosomes, initiation, elongation and termination, the energy cost, the antibiotics and toxins that block each step, and post-translational modifications such as collagen hydroxylation.
- The genetic code is a triplet, degenerate (most amino acids have several codons; variation at the third "wobble" base), unambiguous, non-overlapping and almost universal (mitochondria differ); AUG codes methionine and starts translation; UAA, UAG, UGA are stop codons.
- Aminoacyl-tRNA synthetases attach the right amino acid to each tRNA (uses ATP: the step that ensures accuracy); ribosomes are 70S (30S + 50S) in bacteria and 80S (40S + 60S) in humans; elongation uses GTP, and peptidyl transferase (an rRNA ribozyme in the large subunit) forms peptide bonds; each amino acid added costs about 4 high-energy bonds.
- Inhibitors: aminoglycosides and tetracyclines (30S), chloramphenicol, macrolides, clindamycin, linezolid (50S); diphtheria toxin and Pseudomonas exotoxin A inactivate EF-2 by ADP-ribosylation; ricin and Shiga toxin damage the 60S rRNA.
On this page
The genetic code
A codon is a sequence of three bases on mRNA that specifies one amino acid. With 4 bases, there are 4³ = 64 codons: 61 code for the 20 amino acids and 3 are stop codons.
| Feature | Meaning |
|---|---|
| Triplet | Three bases = one amino acid |
| Degenerate (redundant) | Most amino acids have more than one codon (only methionine and tryptophan have one each); the variation is usually in the third base ("wobble") |
| Unambiguous | Each codon codes for only one amino acid |
| Non-overlapping, commaless | Read continuously in a fixed frame without gaps |
| Universal | The same in almost all organisms (exceptions in mitochondria, where UGA codes for tryptophan) |
- Start codon: AUG (methionine; N-formylmethionine in bacteria).
- Stop codons: UAA, UAG, UGA.
Possible codons = 4 bases ^ 3 positions = 4 × 4 × 4 = 64
Stop codons = 3 → sense codons = 64 − 3 = 61 for 20 amino acids
mRNA: 5′-AUG GCU UAC UGA-3′
AUG = Met (start), GCU = Ala, UAC = Tyr, UGA = stop
→ peptide = Met-Ala-Tyr (3 amino acids)
Minimum coding bases for a 100-amino-acid protein:
100 × 3 = 300 bases + 3 for the stop codon = 303 bases
Wobble hypothesis: the first base of the tRNA anticodon (pairing with the third base of the codon) can pair loosely, so one tRNA can read several codons.
The players
tRNA
- Small RNA with a cloverleaf shape: an anticodon loop (pairs with the mRNA codon) and a 3′ CCA end where the amino acid attaches.
- Contains modified bases (pseudouridine, dihydrouridine).
Aminoacyl-tRNA synthetases
- One enzyme per amino acid; attaches the correct amino acid to its tRNA ("charging") using ATP (→ AMP + PPi: two high-energy bonds).
- Has proofreading activity; it is the main step that ensures accuracy of translation (the ribosome checks only the codon-anticodon match).
Ribosomes
| Feature | Prokaryotic | Eukaryotic |
|---|---|---|
| Whole | 70S | 80S |
| Small subunit | 30S (16S rRNA) | 40S (18S rRNA) |
| Large subunit | 50S (23S, 5S rRNA) | 60S (28S, 5.8S, 5S rRNA) |
S (Svedberg) units measure sedimentation, so they do not add up simply. Mitochondrial ribosomes resemble bacterial ones (explaining some antibiotic toxicities, such as chloramphenicol marrow suppression and aminoglycoside ototoxicity).
The ribosome has three tRNA sites: A (aminoacyl, incoming), P (peptidyl, growing chain), E (exit).
Steps of translation
1. Initiation
- Small subunit binds mRNA: in bacteria at the Shine-Dalgarno sequence; in eukaryotes at the 5′ cap, then scans to the first AUG.
- Initiator tRNA-Met (fMet in bacteria) binds AUG in the P site.
- Large subunit joins; needs initiation factors and GTP.
2. Elongation
- Charged tRNA enters the A site (with elongation factor EF-Tu/eEF1 and GTP).
- Peptidyl transferase forms the peptide bond, moving the chain to the tRNA in the A site. This activity is in the rRNA of the large subunit (23S/28S): a ribozyme.
- Translocation: the ribosome moves one codon along (EF-G/eEF2 and GTP); the empty tRNA leaves from the E site.
The protein is made from the N-terminus to the C-terminus, while the mRNA is read 5′ → 3′.
3. Termination
A stop codon in the A site is recognised by release factors (not tRNA); the chain is released and the ribosome separates.
Many ribosomes can read one mRNA at once: a polysome.
Per amino acid added:
Charging tRNA: ATP → AMP + PPi = 2 high-energy bonds
Entry into the A site: 1 GTP = 1
Translocation: 1 GTP = 1
Total ≈ 4 high-energy bonds per peptide bond
A protein of 100 amino acids has 99 peptide bonds:
99 × 4 = 396 high-energy bonds (plus a little for initiation)
Inhibitors of protein synthesis
| Inhibitor | Target | Notes |
|---|---|---|
| Aminoglycosides (streptomycin, gentamicin) | 30S | Misreading of mRNA; block initiation |
| Tetracyclines | 30S | Block tRNA binding to the A site |
| Chloramphenicol | 50S | Inhibits peptidyl transferase |
| Macrolides (erythromycin) | 50S | Block translocation |
| Clindamycin | 50S | Blocks translocation |
| Linezolid | 50S | Blocks formation of the initiation complex |
| Puromycin | Both (tRNA mimic) | Premature chain termination |
| Cycloheximide | 80S (eukaryotic) | Laboratory use |
| Diphtheria toxin, Pseudomonas exotoxin A | eEF2 | ADP-ribosylation of eEF2 stops translocation; one toxin molecule can kill a cell |
| Ricin (castor bean), Shiga toxin, Shiga-like toxin (E. coli O157) | 60S rRNA (28S) | Remove an adenine from rRNA, stopping elongation; Shiga-like toxin causes haemolytic uraemic syndrome |
Post-translational modification and targeting
- Proteolytic cleavage: removal of signal peptides; prohormones activated (proinsulin → insulin + C-peptide); zymogens activated (trypsinogen → trypsin).
- Glycosylation (in the ER and Golgi), phosphorylation (kinases: regulation), acetylation, methylation, γ-carboxylation of clotting factors II, VII, IX, X (vitamin K-dependent; blocked by warfarin).
- Hydroxylation of proline and lysine in collagen needs vitamin C (deficiency: scurvy, with bleeding gums and poor wound healing).
- Disulfide bonds, folding with chaperones (heat shock proteins). Misfolding causes disease (prion disease, amyloidosis, Alzheimer disease).
- Ubiquitination: marks proteins for breakdown in the proteasome (target of bortezomib in myeloma).
Targeting:
- Proteins made on free ribosomes stay in the cytosol or go to the nucleus, mitochondria or peroxisomes.
- Proteins made on the rough ER (with an N-terminal signal sequence) are secreted, placed in membranes or sent to lysosomes.
- Lysosomal enzymes are tagged with mannose-6-phosphate in the Golgi. I-cell disease (mucolipidosis II): failure to add this tag, so enzymes are secreted into the blood instead of reaching lysosomes; coarse features, developmental delay, death in early childhood.
Expect: features of the genetic code (degenerate, unambiguous, universal, wobble), start and stop codons, role of aminoacyl-tRNA synthetase, 70S vs 80S ribosomes and subunits, peptidyl transferase as a ribozyme, energy use (ATP for charging, GTP for elongation), antibiotics and toxins with their targets (diphtheria toxin and eEF2, ricin and 60S), post-translational modifications (vitamin C in collagen, vitamin K carboxylation) and I-cell disease.
Quick revision
- 64 codons: 61 sense, 3 stop (UAA, UAG, UGA); start AUG.
- Code: triplet, degenerate, unambiguous, non-overlapping, nearly universal.
- Charging: aminoacyl-tRNA synthetase + ATP (ensures accuracy).
- 70S (30S + 50S) vs 80S (40S + 60S).
- Peptidyl transferase is rRNA (ribozyme); elongation uses GTP; about 4 high-energy bonds per amino acid.
- 30S: aminoglycosides, tetracyclines. 50S: chloramphenicol, macrolides, clindamycin, linezolid.
- Diphtheria toxin: ADP-ribosylates eEF2. Ricin, Shiga toxin: 60S rRNA.
- I-cell disease: no mannose-6-phosphate tag.
Written by The House Job from standard biochemistry references (Lippincott Illustrated Reviews: Biochemistry).
FAQ
Which codon starts translation?
AUG, which codes for methionine.
What are the stop codons?
UAA, UAG and UGA.
What does it mean that the genetic code is degenerate?
Most amino acids are coded by more than one codon, usually differing in the third base.
How does diphtheria toxin stop protein synthesis?
It ADP-ribosylates elongation factor 2 (eEF2), blocking translocation of the ribosome.