15.3 Stages of translation: initiation, elongation, and termination

Translation converts the information encoded in mRNA into a polypeptide chain. This is the final step of the central dogma, and understanding its three stages (initiation, elongation, termination) is essential for everything from gene expression regulation to understanding how antibiotics target bacterial ribosomes.

Prokaryotes and eukaryotes share the core translation machinery, but they differ in ribosome size, initiation strategy, and the specific factors involved. Those differences matter clinically and show up frequently on exams.

Translation Initiation

Initiation is all about assembling a ribosome on the mRNA with the initiator tRNA positioned at the start codon (AUG). This is the most regulated stage of translation and the step where prokaryotes and eukaryotes diverge the most.

Prokaryotic Initiation

1. The 30S ribosomal subunit recognizes and binds the Shine-Dalgarno sequence, a purine-rich region located ~5–10 nucleotides upstream of the AUG start codon on the mRNA. This base-pairs with the 16S rRNA in the 30S subunit, positioning the ribosome directly over the start codon.
2. Three initiation factors (IF1, IF2, IF3) and the initiator tRNA (fMet-tRNA$^{\text{fMet}}$) associate with the 30S subunit to form the 30S initiation complex. IF2 is a GTPase that escorts the initiator tRNA to the P site. IF3 prevents premature joining of the 50S subunit, and IF1 blocks the A site.
3. The 50S ribosomal subunit joins, GTP is hydrolyzed by IF2, and the initiation factors are released. This produces the complete 70S initiation complex, with the initiator tRNA seated in the P site, ready for elongation.

Note that prokaryotes use formyl-methionine (fMet) as their first amino acid, not regular methionine.

Eukaryotic Initiation

Eukaryotic initiation is more elaborate and involves many more initiation factors (designated "eIF"). Here's how it unfolds:

1. Formation of the 43S pre-initiation complex:

   • The 40S ribosomal subunit associates with eIF1, eIF1A, eIF3, and eIF5.
   • A ternary complex of eIF2·GTP·Met-tRNA$_i^{\text{Met}}$ joins the 40S subunit, delivering the initiator tRNA.

2. mRNA recruitment and scanning:

   • The eIF4F complex (composed of eIF4E, eIF4A, and eIF4G) binds the 5' cap of the mRNA. eIF4E recognizes the cap, eIF4A is an RNA helicase that unwinds secondary structure, and eIF4G serves as a scaffold.
   • The 43S pre-initiation complex is recruited to the capped mRNA, forming the 48S initiation complex.
   • The 48S complex then scans along the mRNA in the 5'→3' direction until it encounters the first AUG in a favorable sequence context (the Kozak sequence: 5'-GCCRCCAUGG-3', where R = purine).

3. 60S subunit joining:

   • Once the start codon is recognized, eIF5 stimulates GTP hydrolysis by eIF2, triggering release of most initiation factors.
   • The 60S subunit joins to form the complete 80S initiation complex, and elongation can begin.

Translation Elongation

Once the initiation complex is assembled with the initiator tRNA in the P site, the elongation cycle adds amino acids one at a time to the growing polypeptide. Each cycle has three steps.

The Elongation Cycle

Step 1: Aminoacyl-tRNA delivery to the A site

• Elongation factor EF-Tu (eEF1A in eukaryotes) binds an aminoacyl-tRNA along with GTP, forming a ternary complex.
• This complex enters the A site of the ribosome. If the tRNA anticodon correctly base-pairs with the mRNA codon, GTP hydrolysis is triggered, EF-Tu is released, and the aminoacyl-tRNA is accommodated into the A site.
• Incorrect codon-anticodon pairing leads to rejection before GTP hydrolysis, which is how the ribosome maintains translational fidelity.

Step 2: Peptide bond formation

• The peptidyl transferase center, located in the large subunit (50S in prokaryotes, 60S in eukaryotes), catalyzes peptide bond formation. This is a ribozyme activity: the catalysis is performed by the 23S rRNA (or 28S rRNA in eukaryotes), not by a protein enzyme.
• The growing polypeptide attached to the P-site tRNA is transferred to the amino acid on the A-site tRNA, forming a new peptide bond. After this reaction, the P-site tRNA is deacylated (empty) and the A-site tRNA carries the growing chain.

Step 3: Translocation

• Elongation factor EF-G (eEF2 in eukaryotes) binds the ribosome and hydrolyzes GTP, driving the ribosome to shift exactly one codon (3 nucleotides) in the 3' direction along the mRNA.
• The peptidyl-tRNA moves from the A site → P site. The deacylated tRNA moves from the P site → E site (exit site), where it dissociates.
• The A site is now empty and ready to accept the next aminoacyl-tRNA. The cycle repeats, adding roughly 15–20 amino acids per second in prokaryotes (slower in eukaryotes).

Translation Termination

Termination occurs when the ribosome encounters a stop codon and no corresponding tRNA exists to fill the A site.

Termination and Polypeptide Release

1. Stop codon recognition: When one of the three stop codons (UAA, UAG, or UGA) enters the A site, no aminoacyl-tRNA can bind. Instead, release factors recognize the stop codon. In prokaryotes, RF1 recognizes UAA and UAG, while RF2 recognizes UAA and UGA. In eukaryotes, a single factor, eRF1, recognizes all three stop codons.

2. Polypeptide release: The release factor triggers hydrolysis of the ester bond linking the polypeptide to the P-site tRNA. The completed polypeptide is released from the ribosome. In prokaryotes, RF3 (a GTPase) then promotes release of RF1/RF2 from the ribosome.

3. Ribosome recycling: The ribosomal subunits, mRNA, and deacylated tRNA must be separated so they can be reused.

   • In prokaryotes, the ribosome recycling factor (RRF) works with EF-G to split the 70S ribosome back into 30S and 50S subunits. IF3 then binds the 30S subunit to prevent reassociation.
   • In eukaryotes, the recycling mechanism is less well characterized, but involves factors including ABCE1, an ATPase that promotes subunit dissociation.

Prokaryotic vs. Eukaryotic Translation

The fundamental logic of translation is conserved across all life: ribosomes read mRNA codons, tRNAs deliver amino acids, and the same three stages occur in order. The genetic code itself is nearly universal (with minor variations in mitochondria and a few organisms).

The differences, however, are significant:

The fact that prokaryotic and eukaryotic ribosomes differ in structure is why many antibiotics (e.g., chloramphenicol, tetracycline, erythromycin) can target bacterial 70S ribosomes without affecting human 80S ribosomes. This is a direct clinical application of the differences covered here.