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

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:

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

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

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