Translation in Eukaryotes

Key Components Required

Stage 1: Initiation

Steps:

• The 80S ribosome must first dissociate into its 40S and 60S subunits.
• Initiation factors eIF-1, eIF-1A, and eIF-3 bind the newly free 40S subunit and prevent premature reassociation with 60S.

• eIF-2-GTP binds the special initiator Met-tRNA (Met-tRNA_i) to form a ternary complex.
• This ternary complex joins the 40S subunit (along with eIF-1, eIF-1A, eIF-3, and eIF-5) to form the 43S preinitiation complex.
• Note: In eukaryotes, the initiator tRNA carries unmodified methionine (Met) - not formylmethionine (fMet) as in prokaryotes.

• The mRNA is recognized through its 5' cap structure (m7G cap).
• eIF-4E (cap-binding protein) binds the 5' cap.
• eIF-4G (scaffold protein) and eIF-4A (an ATP-dependent RNA helicase) join eIF-4E to form the eIF-4F complex.
• Poly-A binding proteins (PABPs) interact with the poly-A tail and bind to eIF-4G, causing circularization of the mRNA - this likely prevents use of incompletely processed mRNAs.
• The 43S complex binds near the 5' cap and scans 5'→3' along the mRNA (this scanning requires ATP hydrolysis by eIF-4A) until it finds the first AUG codon in a favorable Kozak sequence context.
• This produces the 48S initiation complex.

Cap-independent initiation: Some mRNAs (e.g., viral mRNAs) use an Internal Ribosome Entry Site (IRES) that allows the 40S subunit to bind directly near the start codon without scanning.

• eIF-5 triggers GTP hydrolysis on eIF-2, releasing eIF-2-GDP and several other eIFs.
• The large 60S subunit then joins with the help of eIF-5B-GTP, forming the complete 80S initiation complex.
• The initiator Met-tRNA now occupies the P site of the ribosome, and the A site is empty and ready.

Stage 2: Elongation

• A (Aminoacyl) site - accepts incoming aa-tRNA
• P (Peptidyl) site - holds the growing polypeptide chain
• E (Exit) site - releases the discharged tRNA

Steps:

• eEF-1A-GTP (equivalent to prokaryotic EF-Tu) delivers the correct aminoacyl-tRNA to the A site.
• Codon-anticodon recognition triggers GTP hydrolysis; eEF-1A-GDP is released.
• eEF-1B (equivalent to EF-Ts) exchanges GDP for GTP to regenerate active eEF-1A.

• The peptidyl transferase activity of the 23S rRNA in the 60S subunit (a ribozyme activity) catalyzes formation of a peptide bond between the amino acid in the A site and the growing chain in the P site.
• The chain is transferred from P-site tRNA to the A-site tRNA; the P-site tRNA is now uncharged.

• eEF-2-GTP (equivalent to prokaryotic EF-G) catalyzes movement of the ribosome one codon (3 nucleotides) in the 3' direction.
• The peptidyl-tRNA moves from A→P site, the empty tRNA moves from P→E site (and exits), and the A site is now empty for the next codon.
• GTP is hydrolyzed in this step.

Stage 3: Termination

• When a stop codon (UAA, UAG, or UGA) enters the A site, no tRNA recognizes it.
• Instead, eRF-1 (a eukaryotic release factor that mimics a tRNA structure) recognizes all three stop codons.
• eRF-3-GTP works with eRF-1 to stimulate release.

• eRF-1 triggers the peptidyl transferase to add water instead of an amino acid, hydrolyzing the bond between the polypeptide and the tRNA.
• The completed polypeptide is released from the ribosome.

• The 80S ribosome dissociates into 40S and 60S subunits, which can be reused.

Polysomes (Polyribosomes)

Co- and Post-Translational Modifications

• Trimming - removal of signal peptides or precursor sequences (e.g., insulin, procollagen)
• Phosphorylation - on Ser, Thr, or Tyr residues by protein kinases
• Glycosylation - N-linked (in RER) or O-linked (in Golgi) sugars added
• Acetylation, Hydroxylation, Ubiquitination, and more

Comparison: Eukaryotic vs Prokaryotic Translation

The Wobble Hypothesis

Background - Why Wobble is Needed

Core Concept

• Positions 1 and 2 of the codon (5' end of codon) follow strict, traditional Watson-Crick base pairing with positions 3 and 2 of the anticodon:
  • A pairs with U
  • G pairs with C
  • U pairs with A
  • C pairs with G
• Position 3 of the codon (3' end = the "wobble position") can form non-traditional, flexible pairings with the 5' base of the anticodon (position 1 of anticodon). This flexibility is called wobble.

Wobble Base Pairings

Inosine (I) is a modified base formed by deamination of adenine (its base is hypoxanthine). It is the most promiscuous wobble base - a tRNA with inosine at the 5' anticodon position can read three different codons.

Significance of Wobble

1. Fewer tRNAs needed - 61 tRNAs are NOT required for 61 codons; ~40-45 tRNAs suffice.
2. Explains degeneracy of the genetic code - Multiple codons can specify the same amino acid, and wobble explains how one tRNA handles this.
3. Synonymous codons often differ only at the 3rd position - This is the "third base degeneracy" or "wobble position" seen throughout the genetic code (e.g., Gly: GGU, GGC, GGA, GGG - one tRNA with anticodon CCC can read GGG, and one with anticodon ICC can read GGU, GGC, and GGA).
4. Inosine-containing tRNAs are especially efficient - A single tRNA with inosine at the wobble position can decode three codons.

Simple Summary

mRNA Codon:      5' - N1 - N2 - N3(wobble) - 3'
                      |    |    |
tRNA Anticodon:  3' - N1' - N2' - N3'(wobble) - 5'

Positions 1 & 2 → Strict Watson-Crick pairing
Position 3 (3' of codon / 5' of anticodon) → Flexible "wobble" pairing