Translation and Protein Synthesis

The genetic code, translation process, and protein synthesis

🔤 Translation and Protein Synthesis

The Genetic Code

Codon: Three-nucleotide sequence in mRNA that codes for amino acid

Key features:

  • 64 codons total (4³ = 64)
  • 61 code for amino acids
  • 3 are stop codons: UAA, UAG, UGA
  • 1 start codon: AUG (also codes for methionine)

Properties:

  1. Redundant (degenerate):

    • Multiple codons for most amino acids
    • Example: Leucine has 6 codons

  2. Unambiguous:

    • Each codon specifies only ONE amino acid

  3. Nearly universal:

    • Same in almost all organisms
    • Strong evidence for common ancestry

  4. Non-overlapping:

    • Read in groups of 3, no overlap
    • Frame matters (reading frame)

tRNA Structure and Function

Structure:

  • Cloverleaf shape (2D), L-shape (3D)
  • Anticodon: three bases that pair with mRNA codon
  • Amino acid attachment site: 3' end

Function:

  • Brings correct amino acid to ribosome
  • Anticodon pairs with codon (complementary, antiparallel)

Aminoacyl-tRNA synthetase:

  • Enzyme that attaches amino acid to correct tRNA
  • 20 different synthetases (one per amino acid)
  • Ensures accuracy

Ribosome Structure

Two subunits:

  • Small subunit: binds mRNA
  • Large subunit: catalyzes peptide bond formation

Three binding sites:

  • A site (aminoacyl): incoming tRNA
  • P site (peptidyl): tRNA with growing chain
  • E site (exit): empty tRNA leaves

Ribosomal RNA (rRNA):

  • Catalytic component
  • Ribozyme: RNA with enzymatic activity
  • Forms peptide bonds

Translation Process

1. Initiation

Prokaryotes:

  • Small ribosomal subunit binds mRNA at Shine-Dalgarno sequence
  • Initiator tRNA (fMet-tRNA) binds start codon (AUG)
  • Large subunit joins
  • Initiation factors help

Eukaryotes:

  • Small subunit binds 5' cap
  • Scans for start codon (AUG)
  • Initiator tRNA (Met-tRNA) binds
  • Large subunit joins

2. Elongation

Three steps (repeating):

  1. Codon recognition:

    • Aminoacyl-tRNA enters A site
    • Anticodon pairs with codon

  2. Peptide bond formation:

    • rRNA catalyzes peptide bond
    • Amino acid transferred from P site to A site
    • Growing chain now on A-site tRNA

  3. Translocation:

    • Ribosome moves 3 nucleotides (one codon)
    • tRNA in A site → P site
    • tRNA in P site → E site → exits
    • A site now empty for next tRNA
    • Requires GTP and elongation factors

3. Termination

  • Stop codon enters A site (UAA, UAG, or UGA)
  • Release factors bind (no tRNA for stop codons)
  • Polypeptide released
  • Ribosomal subunits dissociate

Polyribosomes (Polysomes)

  • Multiple ribosomes on one mRNA
  • Simultaneous translation
  • Increases protein production efficiency

Mutations

Point Mutations

Silent mutation:

  • Changes codon but NOT amino acid (due to redundancy)
  • Usually no effect

Missense mutation:

  • Changes codon → different amino acid
  • May affect protein function
  • Example: sickle cell (Glu → Val)

Nonsense mutation:

  • Changes codon → stop codon
  • Premature termination
  • Nonfunctional protein (usually)

Frameshift Mutations

Insertion or deletion of nucleotides (not multiple of 3)

  • Shifts reading frame
  • All downstream codons changed
  • Usually severe effects

Post-Translational Modifications

After translation, proteins may be modified:

  • Cleaving signal sequences
  • Adding chemical groups (phosphorylation, acetylation)
  • Adding sugars (glycosylation)
  • Folding with chaperones
  • Forming disulfide bonds

Key Concepts

  1. Genetic code: 64 codons, 61 for amino acids, 3 stop, 1 start (AUG)
  2. tRNA: brings amino acids, anticodon pairs with codon
  3. Ribosome: catalyzes peptide bonds (rRNA is ribozyme)
  4. Three sites: A (incoming), P (peptide), E (exit)
  5. Elongation: codon recognition → peptide bond → translocation
  6. Stop codons: UAA, UAG, UGA (no tRNA, release factors bind)
  7. Mutations: silent, missense, nonsense, frameshift

📚 Practice Problems

1Problem 1hard

❓ Question:

Describe the three stages of translation: (a) initiation - including the role of the ribosome, mRNA, and initiator tRNA, (b) elongation - including the roles of aminoacyl-tRNA, peptidyl transferase, and translocation, and (c) termination - including release factors.

💡 Show Solution

Translation - Protein Synthesis:

Overview: mRNA → Protein (requires ribosomes, tRNAs, amino acids, energy)

(a) Initiation:

Goal: Assemble ribosome on mRNA at start codon

Components needed:

  • mRNA with 5' cap and start codon (AUG)
  • Small ribosomal subunit (40S in eukaryotes)
  • Large ribosomal subunit (60S in eukaryotes)
  • Initiator tRNA (Met-tRNA^Met)
  • Initiation factors (IF1, IF2, IF3 in prokaryotes; eIFs in eukaryotes)
  • GTP

Step-by-step (Eukaryotes):

Step 1: Small subunit binds to 5' cap of mRNA

  • With help of initiation factors
  • Scans along mRNA for start codon (5' → 3' direction)

Step 2: Small subunit finds AUG

  • Kozak sequence helps identify correct AUG
  • Usually first AUG after 5' cap

Step 3: Initiator tRNA (with Met) binds

  • Anticodon (UAC) pairs with start codon (AUG)
  • Located in P site of ribosome
  • GTP hydrolysis provides energy

Step 4: Large subunit joins

  • Creates complete 80S ribosome
  • Forms three binding sites:
    • A site (aminoacyl-tRNA): incoming tRNA
    • P site (peptidyl-tRNA): holds growing chain
    • E site (exit): departing tRNA

Ribosome structure after initiation:

        Large subunit (60S)
    [E site][P site][A site]
           [Met-tRNA]  [empty]
    ————————————————————————————
        5'...AUG NNN NNN...3' (mRNA)
        Small subunit (40S)

(b) Elongation:

Goal: Add amino acids one by one to growing polypeptide

Cycle repeats for each codon:

Step 1: Aminoacyl-tRNA binding (Codon Recognition)

  • Aminoacyl-tRNA (charged tRNA with amino acid) enters A site
  • Codon-anticodon pairing checked
  • EF-Tu (elongation factor) escorts tRNA with GTP
  • Correct pairing → GTP hydrolysis → EF-Tu released
  • Wrong pairing → tRNA rejected (proofreading!)

Step 2: Peptide bond formation

  • Peptidyl transferase (ribozyme in large subunit rRNA!)
  • Catalyzes peptide bond between:
    • Amino acid in P site (attached to growing chain)
    • Amino acid in A site (new amino acid)
  • Nucleophilic attack: amino group of A-site aa on carbonyl of P-site aa
  • Growing polypeptide now attached to A-site tRNA
  • P-site tRNA now "deacylated" (no amino acid)

Chemical reaction:

P site: tRNA—[Met-Arg-Tyr-]
                    +
A site: tRNA—[Ala]
        ↓ peptidyl transferase
P site: tRNA (empty)
A site: tRNA—[Met-Arg-Tyr-Ala]

Step 3: Translocation

  • EF-G (elongation factor G) with GTP binds
  • Ribosome moves exactly 3 nucleotides (1 codon) in 5' → 3' direction
  • tRNA movements:
    • A-site tRNA (with polypeptide) → P site
    • P-site tRNA (empty) → E site
    • E-site tRNA → released
    • A site now empty for next aminoacyl-tRNA
  • GTP hydrolysis powers movement

After one cycle:

    [E site][P site][A site]
     [empty][tRNA—chain][empty]
    ————————————————————————————
    5'...AUG ARG TYR ALA SER...3'
              (next codon ready)

Elongation repeats until stop codon reaches A site

Speed: ~20 amino acids/second in eukaryotes, ~60/second in prokaryotes!

(c) Termination:

Goal: Release completed polypeptide when stop codon reached

Stop codons: UAA, UAG, UGA (no tRNA matches these!)

Step 1: Recognition

  • Stop codon enters A site
  • No tRNA with matching anticodon
  • Release factor (RF) recognizes stop codon
    • Prokaryotes: RF1 (UAA, UAG) or RF2 (UAA, UGA)
    • Eukaryotes: eRF1 (all three stop codons)

Step 2: Hydrolysis

  • Release factor binds in A site (mimics tRNA structure!)
  • Activates peptidyl transferase
  • Instead of forming peptide bond, hydrolyzes bond between:
    • Polypeptide and tRNA in P site
  • Adds H₂O instead of amino acid
  • Polypeptide released with C-terminus carboxyl group (-COOH)

Chemical reaction:

tRNA—[polypeptide] + H₂O → tRNA + polypeptide-COOH

Step 3: Ribosome dissociation

  • RF3 (prokaryotes) or eRF3 (eukaryotes) with GTP
  • GTP hydrolysis
  • Ribosomal subunits separate
  • mRNA released
  • Deacylated tRNA released
  • Components recycled for next round

Post-translational processing:

  • Initial Met often removed
  • Protein folding (chaperones help)
  • Chemical modifications:
    • Phosphorylation
    • Glycosylation
    • Acetylation
    • Disulfide bonds
  • Protein targeting to destination

Summary Table:

| Stage | Key Events | Factors | Energy | |-------|-----------|---------|--------| | Initiation | Ribosome assembly at AUG | IFs | GTP | | Elongation | Amino acid addition (cycle) | EF-Tu, EF-G | GTP (2 per aa) | | Termination | Release at stop codon | RFs | GTP |

Energy cost:

  • ~4 ATP equivalents per amino acid:
    • 2 GTP for elongation (EF-Tu + EF-G)
    • 1 ATP to charge tRNA (aminoacyl-tRNA synthetase)
    • 1 GTP for initiation/termination (amortized)

Accuracy:

  • Codon-anticodon pairing checked twice
  • Error rate: ~1 in 10,000 amino acids
  • Proofreading by aminoacyl-tRNA synthetases
  • Induced fit mechanism

Translation: Initiation (start) → Elongation (add aa) → Termination (stop)\boxed{\text{Translation: Initiation (start) → Elongation (add aa) → Termination (stop)}}

Polyribosome (Polysome):

  • Multiple ribosomes on same mRNA simultaneously
  • Increases protein production efficiency
  • Each ribosome at different position
  • Makes many copies of same protein at once
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