Table of Contents
- Introduction
- Exam Information
- Ribosomes
- The Genetic Code
- Transfer RNA (tRNA)
- The Wobble Hypothesis
- Translation: Overview
- Initiation of Translation
- Elongation
- Termination
- Post-Translational Modifications
- Protein Targeting After Translation
- Conclusion and Key Takeaways
1. Introduction
Protein synthesis — also called translation — is the process by which the nucleotide sequence of a messenger RNA (mRNA) is decoded into a specific sequence of amino acids to form a protein. It is the final step of gene expression, following DNA replication and transcription.
The central dogma of molecular biology states:
DNA → (Transcription) → mRNA → (Translation) → Protein
This lecture focuses entirely on the translation step, covering:
- The molecular machines involved (ribosomes, tRNAs)
- The genetic code that links nucleotide triplets to amino acids
- The three stages of translation: initiation, elongation, and termination
- What happens to newly synthesized proteins (post-translational modifications and protein targeting)
2. Exam Information
This section summarises the examination rules for Biochemistry Module 2 at the University of Bologna. Understanding the grading criteria can help you prioritise your study efforts.
Format
- The final written exam covers both Chemistry and Biochemistry, each allocated 25 minutes (total 50 minutes).
- Question types include: true/false, single-choice (3 options), and open questions.
Scoring
| Module | Max Points |
|---|---|
| Chemistry | 16.50 |
| Biochemistry | 16.50 |
| Total | 33.00 |
- Passing threshold: at least 9 points out of 16.50 in each module.
- The final grade (≥ 18/30) is obtained by summing both module scores and rounding to the nearest integer.
- A score > 30.5 combined earns 30 cum laude (30L).
- A score of 8.5–8.9 in one module (below 9 but above 8.5) still results in a final grade of 18/30.
- Once the Chemistry module score is accepted, it must not be repeated.
Biochemistry Exam Breakdown
| Question Type | Number | Points Each |
|---|---|---|
| True/False | 8 | 0.40 |
| Single Choice (3 options) | 10 | 0.85 |
| Open Question 1 | 1 | 2.00 |
| Open Question 2 | 1 | 2.80 |
3. Ribosomes
Ribosomes are the molecular machines responsible for translating the mRNA code into protein. They are present in all living cells and are composed of ribosomal RNA (rRNA) and proteins.
3.1 Functions of Ribosomes
Ribosomes perform several essential tasks during translation:
- Bind mRNA — the ribosome positions itself on the mRNA molecule to read the codons.
- Provide sites for aminoacyl-tRNA — three sites exist within the ribosome:
- A site (Aminoacyl site): accepts the incoming aminoacyl-tRNA (carrying a new amino acid).
- P site (Peptidyl site): holds the tRNA carrying the growing polypeptide chain.
- E site (Exit site): holds the discharged (empty) tRNA just before it leaves the ribosome.
- Facilitate interactions with initiation factors — non-ribosomal proteins are needed to start translation.
- Catalyze peptide bond formation — via the peptidyl transferase center (an intrinsic rRNA activity).
- Translocate the peptidyl-tRNA — move the growing chain and mRNA together, one codon at a time in the 5’→3’ direction.
3.2 Structure: Prokaryotic vs. Eukaryotic Ribosomes
Ribosomes are composed of two unequal subunits — a large and a small subunit — that assemble around the mRNA during translation. They are measured in Svedberg units (S), a measure of sedimentation rate.
Prokaryotic Ribosome (70S) — Example: E. coli
| Feature | Detail |
|---|---|
| Overall size | 70S (diameter ≈ 25 nm, mass ≈ 2520 kD) |
| Small subunit | 30S — 21 proteins + 16S rRNA (~930 kD) |
| Large subunit | 50S — 31 proteins + 23S rRNA + 5S rRNA (~1590 kD) |
| Dissociation | Occurs at [Mg²⁺] < 1 mM |
Key fact: Ribosomes are primarily made of RNA (~2/3 by mass). A single E. coli cell contains approximately 20,000 ribosomes, representing about 20% of total cellular mass.
Eukaryotic Ribosome (80S)
| Feature | Detail |
|---|---|
| Overall size | 80S |
| Small subunit | 40S — 30 proteins + 18S rRNA |
| Large subunit | 60S — 45 proteins + 28S rRNA + 5.8S rRNA + 5S rRNA |
Note on organellar ribosomes: Ribosomes found in mitochondria and chloroplasts are similar to prokaryotic 70S ribosomes, consistent with the endosymbiotic theory — the idea that these organelles evolved from free-living bacteria engulfed by ancestral eukaryotic cells.
Despite differences in size between prokaryotic and eukaryotic ribosomes, many structural and functional properties are conserved — this is why some antibiotics that target the 70S prokaryotic ribosome do not affect the 80S eukaryotic ribosome.
4. The Genetic Code
4.1 From DNA to Protein: The Two Essential Components
To translate a 4-letter nucleotide alphabet into a 20-letter amino acid alphabet, two things are required:
-
A Genetic Code: A system of triplet codons (3-nucleotide sequences) where each triplet specifies one amino acid. Using 4 bases in groups of 3 gives 4³ = 64 possible codons.
-
A Translator molecule: This is transfer RNA (tRNA), which physically bridges the codon sequence on the mRNA and the specific amino acid it encodes. Each tRNA carries a specific amino acid and possesses an anticodoncomplementary to the corresponding mRNA codon.
Historical context: The interpretation of the genetic code was awarded the Nobel Prize in Physiology or Medicine in 1968 to:
- Robert W. Holley (Cornell University) — structure of tRNA
- Har Gobind Khorana (University of Wisconsin) — chemical synthesis of nucleotides to decipher codons
- Marshall W. Nirenberg (National Institutes of Health) — cell-free translation experiments
4.2 Codons and the Triplet Code
- With 4 nucleotides (U, C, A, G in RNA) and triplet codons: 4³ = 64 codons
- Of these 64 codons:
- 61 codons specify the 20 amino acids (the sense codons)
- 3 codons are stop codons (also called nonsense codons): UAA, UAG, UGA — these do not code for an amino acid; they signal the end of translation.
The code is non-overlapping: each nucleotide belongs to only one codon. The ribosome reads mRNA in a sequential, non-overlapping fashion in the 5’→3’ direction.
Codon–Anticodon Interaction
- The mRNA codon is read by the anticodon of the tRNA in an antiparallel fashion.
- For example: mRNA codon 5’-AUG-3’ pairs with tRNA anticodon 3’-UAC-5’.
- It is the tRNA, not the mRNA, that directly recognizes the amino acid through the action of aminoacyl-tRNA synthetases.
4.3 Characteristics of the Genetic Code
The genetic code has three key properties:
| Property | Meaning |
|---|---|
| Degeneracy | More than one codon can specify the same amino acid (e.g., leucine is encoded by 6 codons) |
| Non-ambiguity | Each codon specifies only one amino acid (never two different amino acids) |
| Universality | The same codons encode the same amino acids in virtually all organisms, with minor exceptions (notably in mitochondria) |
4.4 Degeneracy of the Genetic Code
Because there are 64 codons but only 20 amino acids (+3 stop signals), the code is degenerate (redundant). The distribution is:
| Number of Codons | Number of Amino Acids | Examples |
|---|---|---|
| 1 | 2 | Methionine (AUG), Tryptophan (UGG) |
| 2 | 9 | Phenylalanine, Tyrosine, Histidine, Glutamine, Asparagine, Lysine, Aspartate, Glutamate, Cysteine |
| 3 | 2 | Isoleucine, STOP |
| 4 | 5 | Valine, Proline, Threonine, Alanine, Glycine |
| 6 | 3 | Leucine, Arginine, Serine |
Important observation: The first two bases of a codon are the most informative — codons for the same amino acid often differ only in the third base. This third base is called the wobble position and corresponds to the first base of the anticodon. Less stringent base-pairing rules apply here.
4.5 Codon Table
The standard codon table (reading mRNA 5’→3’) is organized by the first, second, and third codon positions. Key codons to memorize:
- Start codon: AUG (methionine)
- Stop codons: UAA, UAG, UGA
- Single-codon amino acids: AUG (Met), UGG (Trp) — these have no wobble
- Six-codon amino acids: Leucine (UUA, UUG, CUU, CUC, CUA, CUG), Arginine, Serine
5. Transfer RNA (tRNA)
tRNA molecules are the adaptor molecules that physically link a specific codon on the mRNA to its corresponding amino acid. There are multiple different tRNA species in the cell — at least one for each of the 20 amino acids.
5.1 Structure of tRNA
tRNA molecules are single-stranded RNA that fold into a characteristic cloverleaf secondary structure (2D representation), which folds further into an L-shaped tertiary structure (3D).
The key structural regions are:
| Region | Description |
|---|---|
| 5’ Phosphate | Phosphate group at the 5’ end |
| Acceptor Stem | 7 base pair stem; the amino acid attaches here at the 3’ end |
| D Arm | Stem of 3–4 bp + loop of 5–7 bp; contains dihydrouridine (D) residues |
| Anticodon Arm | Stem of 5 bp ending in a loop that contains the anticodon (the 3-nucleotide sequence complementary to the mRNA codon) |
| T Arm (TψC arm) | Stem of 5 bp + loop; contains pseudouridine (ψ) |
| Variable Region | Region of variable size between the anticodon arm and T arm; not present in all tRNAs |
| CCA Sequence | Universal 3’ end: -C-C-A-OH; the 3’-OH of the terminal adenosine is the site of amino acid attachment |
Key detail: The D arm contains 2 or 3 dihydrouridine (D) residues, and in some tRNAs has only 3 hydrogen-bonded base pairs. The anticodon is read in the 3’→5’ direction (antiparallel to the 5’→3’ mRNA codon).
5.2 Amino Acid Attachment to tRNA
The amino acid is covalently linked to the 3’-OH of the terminal adenosine in the -CCA sequence (the acceptor stem) via a high-energy ester bond. A tRNA carrying its amino acid is called a charged tRNA or aminoacyl-tRNA.
5.3 Aminoacyl-tRNA Synthetases
The enzymes responsible for attaching amino acids to their corresponding tRNAs are the aminoacyl-tRNA synthetases (aaRS). There is (at least) one for each of the 20 amino acids.
Reaction Mechanism: Amino Acid Activation
The reaction occurs in two steps:
Step 1 — Adenylation:
Amino acid + ATP → Aminoacyl-AMP (aminoacyl-adenylate) + PPi
Step 2 — Transfer:
Aminoacyl-AMP + tRNA → Aminoacyl-tRNA + AMP
The hydrolysis of pyrophosphate (PPi) by pyrophosphatase drives the reaction forward irreversibly.
Two Classes of Aminoacyl-tRNA Synthetases
| Class | Attachment Position | Anticodon Recognition |
|---|---|---|
| Class I | 2’-OH of ribose in the terminal A of tRNA | Typically recognizes the anticodon |
| Class II | 3’-OH of ribose in the terminal A of tRNA | Does not typically recognize the anticodon; focuses on other structural features |
Class I synthetases (10 enzymes) include: Arg, Cys, Gln, Glu, Ile, Leu, Met, Trp, Tyr, Val Class II synthetases (10 enzymes) include: Ala, Asn, Asp, Gly, His, Lys, Phe, Pro, Ser, Thr
Specificity of Aminoacyl-tRNA Synthetases
Each synthetase must discriminate correctly at two levels:
- First level: Selecting the correct amino acid (forming the right aminoacyl-adenylate)
- Second level: Selecting the correct tRNA molecule
5.4 Proofreading and Kinetic Bowl Correction
Aminoacyl-tRNA synthetases have a proofreading (editing) mechanism to prevent errors:
Proofreading can occur at two stages:
- If the wrong aminoacyl-adenylate enters the proofreading site, it is hydrolyzed before being transferred.
- If an incorrect amino acid is loaded onto the tRNA, the misacylated aminoacyl-tRNA assumes the wrong conformation and is hydrolyzed.
Kinetic Bowl Correction (Kinetic Proofreading): The tRNA binds to the enzyme by a two-step process:
- The tRNA associates rapidly but dissociates slowly (high affinity)
- The correct tRNA triggers a conformational change that stabilizes its binding to the enzyme
Incorrect tRNAs do not trigger this conformational change and dissociate more readily. This mechanism increases overall fidelity beyond what equilibrium binding alone could achieve.
6. The Wobble Hypothesis
The wobble hypothesis (proposed by Francis Crick) explains how a single tRNA can recognize more than one codon.
- The first two bases of the mRNA codon form standard (canonical) Watson-Crick hydrogen bonds with the 2nd and 3rd bases of the anticodon.
- The third base of the codon (= wobble position) pairs with the first base of the anticodon using non-canonical (wobble) base pairings.
This looser pairing at the wobble position allows one tRNA to read multiple codons (those that differ only in the 3rd position).
Rules of Wobble Pairing
| Anticodon 1st Base (5’) | Codons Recognized (3rd position) | Number |
|---|---|---|
| C | G only | 1 codon |
| A | U only | 1 codon |
| U | A or G | 2 codons |
| G | U or C | 2 codons |
| I (Inosine) | U, C, or A | 3 codons |
Inosine (I) is a modified base derived from hypoxanthine; it is found at the first position of many anticodons and allows the broadest wobble pairing.
Practical implication: To read all 61 sense codons, the cell needs only 32 different tRNA species (not 61), because wobble base pairing allows one tRNA to recognize multiple codons.
Important consequence: Wobble pairing induces faster dissociation of the tRNA from the mRNA after peptide bond formation, which helps maintain the pace of translation.
7. Translation: Overview
Translation occurs in the cytosol of all cell types (and in organelles for mitochondrial/chloroplast proteins).
The Three Sites of the Ribosome
The ribosome has three functional tRNA binding sites:
- A site (Aminoacyl site): Accepts the incoming aminoacyl-tRNA
- P site (Peptidyl site): Holds the tRNA carrying the growing polypeptide
- E site (Exit site): Holds the uncharged (deacylated) tRNA just before it leaves
Three Stages of Translation
- Initiation — Assembly of the ribosome on the mRNA at the start codon (AUG)
- Elongation — Sequential addition of amino acids to the growing chain
- Termination — Release of the finished polypeptide when a stop codon is reached
General rule: A single ribosome reads one mRNA at a time. The ribosome moves along the mRNA in the 5’→3’ direction, and the polypeptide chain grows from its N-terminus to its C-terminus.
8. Initiation of Translation
The Start Codon
- The universal start codon is AUG (codes for methionine).
- Two distinct tRNA species recognize AUG:
- Initiator tRNA — used at the start of translation (occupies the P site)
- Elongator tRNA — used for internal AUG codons (enters the A site)
| Organism Type | First Amino Acid | Notes |
|---|---|---|
| Prokaryotes | N-formyl-methionine (fMet) | Modified methionine; often cleaved from ~50% of final proteins |
| Eukaryotes | Methionine (Met) | Unmodified |
8.1 Initiation in Prokaryotes
Prokaryotic translation initiation relies on several key components and a specific RNA-RNA interaction:
Key Components:
- mRNA with a Shine-Dalgarno sequence (consensus: 5’-AGGAGG-3’) located ~8 nucleotides upstream of the AUG start codon. This sequence base-pairs with the 3’ end of the 16S rRNA in the 30S subunit.
- 30S small subunit
- 50S large subunit
- Initiation Factors: IF-1, IF-2, IF-3
- fMet-tRNA (initiator tRNA) carrying N-formyl-methionine
- GTP
Steps:
- The 30S subunit binds IF-1 and IF-3.
- The 30S subunit binds the mRNA via the Shine-Dalgarno sequence.
- IF-2-GTP binds the 30S subunit and recruits the fMet-tRNAᶠᴹᵉᵗ (which base-pairs with the AUG codon at the P site) → forming the 30S initiation complex.
- The 50S subunit joins the complex, GTP is hydrolyzed, and IF-1, IF-2 (as GDP), and IF-3 dissociate → forming the 70S initiation complex.
- The fMet-tRNA occupies the P site; the A site is empty and ready for the first elongator aminoacyl-tRNA.
8.2 Initiation in Eukaryotes
Eukaryotic initiation is more complex and involves many more initiation factors (eIFs). It is also regulated at multiple levels (important for cell growth control).
Key Features:
- Eukaryotic mRNA is recognized by its 5’ m⁷G cap (7-methylguanosine cap) and 3’ poly(A) tail.
- The mRNA folds into a circular loop structure, mediated by initiation factors, bringing the 5’ cap and 3’ poly(A) tail into proximity.
Steps:
- eIF-2-GTP binds the Met-tRNA (initiator) → forming a ternary complex (eIF2•GTP•Met-tRNAᵢ).
- The ternary complex binds the 40S small subunit (with help from eIF3 and eIF1A) → forming the 43S pre-initiation complex.
- This complex recognizes the eIF-4E (which is bound to the 5’ cap) and eIF4G (which links to the poly(A) tail via PABP) → forming the 48S pre-initiation complex.
- The 40S subunit scans the mRNA in the 5’→3’ direction until the Met-tRNA recognizes the AUG start codon.
- GTP is hydrolyzed; eIFs dissociate; the 60S large subunit joins → forming the 80S initiation complex.
- Translation begins with Met-tRNA in the P site.
9. Elongation
Elongation is the cyclic process by which amino acids are sequentially added to the growing polypeptide chain. Each cycle involves three steps:
9.1 Step 1 — Aminoacyl-tRNA Binding (A Site)
- An elongation factor (EF-Tu in prokaryotes; eEF1A in eukaryotes) forms a ternary complex with aminoacyl-tRNA and GTP.
- This complex delivers the aminoacyl-tRNA to the A site of the ribosome.
- Proofreading: Before GTP hydrolysis, the ribosome verifies correct codon-anticodon pairing. If the pairing is wrong, the aminoacyl-tRNA dissociates before GTP hydrolysis occurs.
- If the match is correct, EF-Tu hydrolyzes GTP → EF-Tu•GDP dissociates from the ribosome, leaving the aminoacyl-tRNA in the A site.
- EF-Tu is recycled: another factor (EF-Ts) exchanges GDP for GTP, regenerating active EF-Tu•GTP.
Note: EF-Tu is extraordinarily abundant — in E. coli, it represents approximately 5% of total cellular proteins.
9.2 Step 2 — Peptide Bond Formation
- Peptidyl transferase catalyzes the formation of the peptide bond between:
- The carboxyl group of the amino acid on the P-site tRNA (the growing chain)
- The free amino group of the incoming amino acid on the A-site tRNA
- After this reaction:
- The P-site tRNA becomes uncharged (deacylated)
- The A-site tRNA now carries the elongated polypeptide chain (peptidyl-tRNA)
Critical insight: Peptidyl transferase activity is NOT due to ribosomal proteins — it is an intrinsic catalytic activity of the 23S rRNA (in prokaryotes) or 28S rRNA (in eukaryotes). The ribosome is therefore a ribozyme. The ‘reaction center’ of the 23S rRNA is among the most highly conserved sequences across all life forms.
The peptidyl transferase center (PTC) is located in the large ribosomal subunit: 50S in prokaryotes, 60S in eukaryotes.
9.3 Step 3 — Translocation
- EF-G (in prokaryotes; eEF2 in eukaryotes) — a translocase — binds at the A site and promotes translocation by hydrolyzing GTP.
- During translocation:
- The uncharged tRNA moves from P site → E site (and then exits the ribosome)
- The peptidyl-tRNA moves from A site → P site
- The mRNA moves exactly one codon (3 nucleotides) in the 5’→3’ direction
- The A site is now free for the next aminoacyl-tRNA
- EF-G has a structural analogy to the EF-Tu•tRNA ternary complex — both occupy the A site, which helps explain how the ribosome alternates between the two.
9.4 Energy Cost of Elongation
Adding each amino acid requires 5 high-energy phosphate bonds in total:
- 2 from ATP — used in amino acid activation by aminoacyl-tRNA synthetase (ATP → AMP + PPi; equivalent to 2 high-energy bonds)
- 3 from GTP — used during elongation:
- 1 GTP hydrolyzed by EF-Tu (delivering aminoacyl-tRNA to A site)
- 1 GTP hydrolyzed by EF-G (translocation)
- 1 additional GTP hydrolysis
GTP hydrolysis primarily drives conformational changes in the elongation factors, rather than directly powering the chemical reaction of peptide bond formation.
10. Termination
Stop Codons
When the ribosome encounters a stop codon (UAA, UAG, or UGA) in the A site, no aminoacyl-tRNA recognizes it. Instead, release factors (RF) bind.
| Organism | Release Factor | Function |
|---|---|---|
| Prokaryotes | RF-1 | Recognizes UAA and UAG |
| Prokaryotes | RF-2 | Recognizes UAA and UGA |
| Prokaryotes | RF-3 | Facilitates release of ribosomal subunits (GTPase) |
| Eukaryotes | eRF1 | Recognizes all three stop codons |
| Eukaryotes | eRF3 | Facilitates release of ribosomal subunits |
Mechanism
- A release factor binds the A site when a stop codon is present.
- The release factor induces a conformational change in the peptidyl transferase center, converting it from a transferase to a hydrolase.
- The peptide chain is cleaved from the P-site tRNA via hydrolysis (water attacks instead of an amino group) → the completed polypeptide is released into the cytosol.
- The ribosome dissociates from the mRNA and the release factors:
- In prokaryotes: IF-3 (an initiation factor) associates with the dissociating subunits; the mRNA and ribosomal units detach.
- The small and large subunits are recycled for new rounds of translation.
11. Post-Translational Modifications
After translation, proteins frequently undergo post-translational modifications (PTMs) that alter their structure, activity, stability, or localization. The major PTMs are:
| Modification | Description | Examples |
|---|---|---|
| Phosphorylation | Addition of a phosphate group (-PO₄) to Serine, Threonine, or Tyrosine | Kinase-mediated signaling |
| Glycosylation | Addition of a sugar (e.g., glucose) to N or O atoms of side chains | Membrane and secreted proteins |
| Lipidation | Addition of a lipid (e.g., fatty acid) to a protein | Membrane anchoring |
| Ubiquitination | Addition of ubiquitin protein to the ε-NH₂ of Lysine side chains | Targets protein for proteasomal degradation |
| Acetylation | Addition of an acetyl group to the N-terminal amino group | Affects protein stability |
| Disulfide bridges | Covalent bond between two Cysteine residues (extracellular only, due to oxidizing environment) | Stabilizes secreted proteins |
| Proteolytic cleavage | N-terminal or C-terminal modifications; removal of signal sequences or propeptides | Insulin maturation |
| Addition of prosthetic groups | Non-protein chemical groups permanently associated with a protein | Heme group in hemoglobin or cytochrome c |
12. Protein Targeting After Translation
Not all newly synthesized proteins remain in the cytosol — many must be directed to specific cellular locations. This is especially critical for membrane proteins and secretory proteins.
Signal Sequences
- These proteins are synthesized with a signal peptide (also called a leader sequence) of approximately 16–26 amino acids at their N-terminus.
- The signal sequence is recognized by the Signal Recognition Particle (SRP), which directs the ribosome to the endoplasmic reticulum (ER) membrane.
- Translation continues as the growing polypeptide is threaded into the ER lumen.
- The signal sequence is usually cleaved by a signal peptidase after translocation.
Protein Sorting Destinations
From the cytosol, newly synthesized proteins can be directed to:
| Destination | Mechanism |
|---|---|
| Cytoplasm | No signal sequence needed; remains soluble in cytosol |
| Nucleus | Nuclear localization signal (NLS) |
| Mitochondria / Chloroplasts | Specific targeting sequences (import post-translationally) |
| Endoplasmic Reticulum | Signal sequence → SRP-mediated co-translational import |
| Golgi Complex | Via vesicular transport from ER |
| Cell Membrane or Secretion | Via the secretory pathway (ER → Golgi → plasma membrane/extracellular) |
13. Conclusion and Key Takeaways
Protein synthesis is a highly coordinated, multi-step process involving dozens of molecular components working together with remarkable accuracy. Here is a summary of the essential concepts:
Summary Table
| Concept | Key Point |
|---|---|
| Ribosome | 70S (prokaryotes) or 80S (eukaryotes); primarily rRNA; three tRNA sites (A, P, E) |
| Genetic Code | Triplet codons; 61 sense + 3 stop; degenerate, non-ambiguous, universal |
| tRNA | Cloverleaf structure; CCA-3’ end; anticodon loop; adaptor between codon and amino acid |
| Aminoacyl-tRNA synthetase | Charges tRNA; two classes; proofreading activity; uses ATP |
| Wobble hypothesis | 3rd codon position has relaxed base-pairing; inosine (I) recognizes 3 codons; 32 tRNAs suffice for 61 codons |
| Initiation | AUG start codon; fMet (prokaryotes) or Met (eukaryotes); Shine-Dalgarno (prokaryotes) or cap-scanning (eukaryotes) |
| Elongation | EF-Tu (A site delivery) → Peptidyl transferase (bond formation) → EF-G (translocation); 5 high-energy bonds/aa |
| Peptidyl transferase | Activity resides in rRNA (ribosome is a ribozyme); located in large subunit |
| Termination | Stop codons (UAA, UAG, UGA); release factors; polypeptide hydrolytically released |
| Post-translational modifications | Phosphorylation, glycosylation, ubiquitination, proteolytic cleavage, etc. |
| Protein targeting | Signal sequences direct proteins to ER, mitochondria, nucleus, etc. |
Connections Between Concepts
DNA
↓ Transcription
mRNA (with 5' cap and 3' poly(A) tail in eukaryotes)
↓
INITIATION: Ribosome + Met-tRNA assembles at AUG
↓
ELONGATION: EF-Tu brings aa-tRNA → Peptidyl transferase forms bond → EF-G translocates
↓ (cycle repeats)
TERMINATION: Stop codon → Release factor → Polypeptide released
↓
POST-TRANSLATIONAL MODIFICATIONS
↓
PROTEIN TARGETING (if signal sequence present)
↓
FUNCTIONAL PROTEIN
Tips for the Exam
- Know the three ribosomal sites (A, P, E) and what occupies each during elongation.
- Be able to describe the energy cost per amino acid incorporated (5 high-energy bonds: 2 ATP-equivalents + 3 GTP).
- Understand the difference between prokaryotic and eukaryotic initiation (Shine-Dalgarno vs. 5’ cap scanning; fMet vs. Met; IFs vs. eIFs).
- Know the three stop codons (UAA, UAG, UGA) and the role of release factors.
- Be able to explain the wobble hypothesis and why only 32 tRNAs are needed for 61 codons.
- Remember that peptidyl transferase activity is a property of the rRNA, not of protein — the ribosome is a ribozyme.
- Know the key post-translational modifications and what they do.
Document prepared from lecture slides — Biochemistry Module 2, Protein Synthesis (RNA Translation), University of Bologna.