🧱 Translation (Protein Synthesis)
Study mRNA translation at ribosomes for CUET Agriculture. Initiation, elongation, termination, tRNA charging and polysome concept covered.
Translation Overview
Translation is the process by which the mRNA sequence is decoded to synthesize a specific polypeptide (protein).
- Occurs at ribosomes in the cytoplasm (or on rough ER for secretory proteins).
- Direction: 5'→3' on mRNA; polypeptide grows from N-terminus → C-terminus.
- Requires: mRNA, tRNAs, ribosomes, amino acids, ATP/GTP, protein factors.
Ribosomes
Ribosomes are the molecular machines of translation. Composed of two subunits — each made of rRNA + proteins.
| Component | Prokaryotes | Eukaryotes |
|---|---|---|
| Whole ribosome | 70S | 80S |
| Large subunit | 50S (23S rRNA + 5S rRNA + ~31 proteins) | 60S (28S + 5.8S + 5S rRNA + ~49 proteins) |
| Small subunit | 30S (16S rRNA + ~21 proteins) | 40S (18S rRNA + ~33 proteins) |
NOTE
S = Svedberg unit (sedimentation coefficient); subunit S values are not additive because they reflect shape and density, not just mass.
Ribosome Sites (A, P, E)
| Site | Name | Function |
|---|---|---|
| A site | Aminoacyl site | Receives incoming aminoacyl-tRNA (aa-tRNA) |
| P site | Peptidyl site | Holds tRNA carrying the growing polypeptide chain |
| E site | Exit site | Holds deacylated (empty) tRNA before it exits the ribosome |
TIP
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Translation Overview
Translation is the process by which the mRNA sequence is decoded to synthesize a specific polypeptide (protein).
- Occurs at ribosomes in the cytoplasm (or on rough ER for secretory proteins).
- Direction: 5'→3' on mRNA; polypeptide grows from N-terminus → C-terminus.
- Requires: mRNA, tRNAs, ribosomes, amino acids, ATP/GTP, protein factors.
Ribosomes
Ribosomes are the molecular machines of translation. Composed of two subunits — each made of rRNA + proteins.
| Component | Prokaryotes | Eukaryotes |
|---|---|---|
| Whole ribosome | 70S | 80S |
| Large subunit | 50S (23S rRNA + 5S rRNA + ~31 proteins) | 60S (28S + 5.8S + 5S rRNA + ~49 proteins) |
| Small subunit | 30S (16S rRNA + ~21 proteins) | 40S (18S rRNA + ~33 proteins) |
NOTE
S = Svedberg unit (sedimentation coefficient); subunit S values are not additive because they reflect shape and density, not just mass.
Ribosome Sites (A, P, E)
| Site | Name | Function |
|---|---|---|
| A site | Aminoacyl site | Receives incoming aminoacyl-tRNA (aa-tRNA) |
| P site | Peptidyl site | Holds tRNA carrying the growing polypeptide chain |
| E site | Exit site | Holds deacylated (empty) tRNA before it exits the ribosome |
TIP
Think of translation as an assembly line: new amino acids enter at A, peptide transfers and chain grows at P, empty tRNA exits at E. Movement is always A→P→E.
Activation of Amino Acids (Aminoacylation)
Before translation can begin, each amino acid must be activated (attached to its cognate tRNA).
- Amino acid + ATP → aminoacyl-AMP + PPi (by aminoacyl-tRNA synthetase).
- Aminoacyl-AMP + tRNA → aminoacyl-tRNA (aa-tRNA) + AMP.
- Net: 2 ATP equivalents consumed per amino acid activation (ATP → AMP = 2 high-energy bonds).
- Each amino acid has its own specific aminoacyl-tRNA synthetase.
- These enzymes ensure fidelity of the genetic code (correct aa matched to correct tRNA).
Steps of Translation
1. Initiation
Prokaryotic initiation:
- Small subunit (30S) + initiation factors (IF-1, IF-2, IF-3) bind mRNA.
- Shine-Dalgarno sequence: purine-rich sequence (~AGGAGG) on mRNA, ~5-10 nt upstream of AUG start codon. Complementary to 3' end of 16S rRNA → positions ribosome at AUG.
- fMet-tRNAᶠᴹᵉᵗ (initiator tRNA; carries N-formyl methionine) binds directly to P site at AUG.
- IF-3 prevents premature association of large subunit.
- GTP hydrolysis → IFs released → 50S subunit joins → 70S initiation complex formed.
Eukaryotic initiation:
- Small subunit (40S) + eIFs (eukaryotic initiation factors) + Met-tRNAᵢᴹᵉᵗ form a pre-initiation complex.
- Complex scans mRNA from 5' cap until it finds AUG.
- Kozak sequence: GCCRCCAUGG (R = purine); context around AUG that determines initiation efficiency.
- 60S subunit joins → 80S initiation complex.
- Met (not fMet) initiates eukaryotic translation.
NOTE
In eukaryotes, the initiator Met is not formylated. In prokaryotes, initiation uses fMet (N-formyl methionine). The formyl group is often removed post-translationally.
2. Elongation
Elongation occurs in a cycle of 3 steps:
Step 1 — Aminoacyl-tRNA delivery (A-site entry):
- EF-Tu·GTP (prokaryotic) / eEF1A·GTP (eukaryotic) delivers aminoacyl-tRNA to A site.
- Codon-anticodon pairing → GTP hydrolysis → EF-Tu released → aa-tRNA accommodated into A site.
Step 2 — Peptide bond formation (Transpeptidation):
- The 23S rRNA (in 50S subunit) catalyzes peptide bond formation — rRNA acts as a ribozyme.
- Peptidyl transferase activity = ribozyme activity of 23S rRNA (proven by H.F. Noller, 1992).
- Polypeptide chain is transferred from P-site tRNA → A-site amino acid.
- P-site tRNA becomes deacylated; A-site tRNA now carries the extended polypeptide.
Step 3 — Translocation:
- EF-G·GTP (prokaryotic) / eEF2·GTP (eukaryotic) facilitates translocation.
- Ribosome moves 3 nucleotides (one codon) in the 5'→3' direction on mRNA.
- A-site tRNA (with polypeptide) → moves to P site.
- P-site tRNA (deacylated) → moves to E site → exits.
- A site is now empty → ready for next aa-tRNA.
- GTP hydrolysis drives translocation.
3. Termination
- Occurs when a stop codon (UAA, UAG, or UGA) enters the A site.
- No tRNA recognizes stop codons.
- Release factors (RF) recognize stop codons:
- RF1 — recognizes UAA and UAG.
- RF2 — recognizes UAA and UGA.
- RF3 — stimulates RF1/RF2 activity (GTPase).
- Eukaryotes have a single eRF1 (recognizes all 3 stop codons) + eRF3.
- RF binds A site → peptidyl transferase cleaves polypeptide from final P-site tRNA → polypeptide released.
- Ribosome dissociates into subunits (aided by RRF — ribosome recycling factor in prokaryotes).
Polysomes (Polyribosomes)
- Multiple ribosomes translating a single mRNA simultaneously.
- Increases efficiency — one mRNA → many polypeptide copies at once.
- In prokaryotes, transcription and translation are coupled (occur simultaneously in cytoplasm).
Post-Translational Modifications
After translation, polypeptides may undergo:
- Cleavage (e.g., removal of signal peptide, fMet, or pro-sequences).
- Glycosylation (addition of sugar groups).
- Phosphorylation, acetylation, ubiquitination.
- Folding assisted by chaperone proteins (e.g., Hsp70).
- Disulfide bond formation.
Antibiotics That Inhibit Translation
Antibiotic inhibition table
| Antibiotic | Target | Mechanism |
|---|---|---|
| Streptomycin | Prokaryotic 30S subunit | Causes misreading of mRNA; inhibits initiation |
| Tetracycline | Prokaryotic 30S subunit | Blocks aminoacyl-tRNA binding to A site |
| Chloramphenicol | Prokaryotic 50S subunit | Inhibits peptidyl transferase (blocks peptide bond formation) |
| Erythromycin | Prokaryotic 50S subunit | Blocks translocation; blocks exit tunnel |
| Linezolid | Prokaryotic 50S subunit | Inhibits initiation (binds 23S rRNA) |
| Cycloheximide | Eukaryotic 60S subunit | Inhibits peptidyl transferase in eukaryotes (NOT prokaryotes) |
| Rifampicin | Prokaryotic RNA polymerase | Inhibits transcription (not translation directly); blocks RNA synthesis |
| Fusidic acid | EF-G | Prevents EF-G dissociation after GTP hydrolysis; blocks translocation |
| Puromycin | Both prokaryotes & eukaryotes | tRNA analog; premature chain termination |
WARNING
Rifampicin acts on RNA polymerase (inhibits transcription), not translation directly — but is often grouped here because it indirectly prevents protein synthesis. Remember: antibiotics targeting 30S or 50S affect prokaryotes; cycloheximide specifically targets eukaryotic 60S.
Key Points to Remember
- Ribosomes: 70S in prokaryotes (30S + 50S); 80S in eukaryotes (40S + 60S). S units are not additive.
- Ribosome sites: A (incoming aa-tRNA), P (growing chain), E (exit).
- Activation: 2 ATP equivalents consumed; aminoacyl-tRNA synthetase ensures fidelity.
- Prokaryotic initiation: Shine-Dalgarno sequence + fMet + IF-1/2/3.
- Eukaryotic initiation: 5' cap scanning + Kozak sequence + Met (not fMet) + eIFs.
- Elongation: EF-Tu (aa-tRNA delivery) → peptide bond (ribozyme: 23S rRNA) → EF-G (translocation); each cycle = 2 GTP.
- Termination: RF1 (UAA/UAG), RF2 (UAA/UGA), RF3 (GTPase); eukaryotes = eRF1 + eRF3.
- Peptidyl transferase = 23S rRNA ribozyme (proven by Noller).
- Antibiotics: streptomycin/tetracycline → 30S; chloramphenicol/erythromycin → 50S; cycloheximide → eukaryotic 60S; rifampicin → RNA polymerase.
Summary Cheat Sheet
| Concept / Topic | Key Details / Explanation |
|---|---|
| Translation | mRNA decoded to synthesize polypeptide at ribosomes in cytoplasm |
| Direction | mRNA read 5'→3'; polypeptide grows N-terminus → C-terminus |
| Prokaryotic ribosome | 70S = 30S (16S rRNA) + 50S (23S + 5S rRNA) |
| Eukaryotic ribosome | 80S = 40S (18S rRNA) + 60S (28S + 5.8S + 5S rRNA) |
| S units | Not additive (reflect shape and density, not just mass) |
| Ribosome A site | Aminoacyl site — receives incoming charged tRNA |
| Ribosome P site | Peptidyl site — holds tRNA with growing polypeptide |
| Ribosome E site | Exit site — holds empty tRNA before release |
| Aminoacylation | Amino acid + ATP → aminoacyl-tRNA; costs 2 ATP equivalents; by aminoacyl-tRNA synthetase |
| Prokaryotic initiation | Shine-Dalgarno sequence (~AGGAGG) on mRNA; fMet-tRNA at P site; IF-1, IF-2, IF-3 |
| Eukaryotic initiation | 5' cap scanning; Kozak sequence (GCCRCCAUGG); Met (not fMet); eIFs |
| Elongation Step 1 | EF-Tu·GTP delivers aa-tRNA to A site (codon-anticodon matching) |
| Elongation Step 2 | Peptide bond formation by 23S rRNA (ribozyme = peptidyl transferase); proven by H.F. Noller (1992) |
| Elongation Step 3 | EF-G·GTP drives translocation — ribosome moves one codon (3 nt) on mRNA |
| Termination | Stop codon (UAA/UAG/UGA) enters A site; no tRNA recognizes it |
| Release factors (prokaryotic) | RF1 → UAA, UAG; RF2 → UAA, UGA; RF3 → GTPase |
| Release factors (eukaryotic) | eRF1 (all 3 stop codons) + eRF3 |
| Polysomes | Multiple ribosomes on single mRNA simultaneously; increases efficiency |
| Coupled transcription-translation | Only in prokaryotes (both in cytoplasm) |
| Post-translational modifications | Cleavage, glycosylation, phosphorylation, acetylation, ubiquitination, folding by chaperones |
| Streptomycin | Inhibits prokaryotic 30S; causes mRNA misreading |
| Tetracycline | Blocks aa-tRNA binding to A site (30S) |
| Chloramphenicol | Inhibits peptidyl transferase (50S) |
| Erythromycin | Blocks translocation (50S) |
| Cycloheximide | Inhibits peptidyl transferase in eukaryotic 60S only |
| Rifampicin | Inhibits prokaryotic RNA polymerase (transcription, not translation) |
| Puromycin | tRNA analog; causes premature chain termination; affects both pro- and eukaryotes |
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