Questions: Translation Initiation: Start Codons and Ribosomal Scanning

Question 1

A eukaryotic mRNA has two AUG codons in its 5' UTR region: the first is in a poor Kozak context, the second is in a strong Kozak context. Which initiation pattern is most likely?

• A. AUG(1) is always used exclusively, because the scanning ribosome initiates at the first AUG it encounters regardless of context
• B. AUG(2) is always used exclusively, because strong Kozak context always overrides position
• C. Most ribosomes initiate at AUG(1), but poor Kozak context allows some to scan past (leaky scanning) and initiate at AUG(2)
• D. Both AUGs are used equally, because eukaryotes use Shine-Dalgarno sequences that can recognize any AUG independently

Answer: (C)

The eukaryotic scanning model generally initiates at the first AUG, but Kozak context modulates efficiency. A strong Kozak context (purine at −3, G at +4) allows nearly all scanning ribosomes to initiate at that AUG. A poor Kozak context allows some ribosomes to scan past — 'leaky scanning' — and initiate at the next AUG instead. Neither AUG always wins: the ratio of initiation events depends on context strength. This mechanism is exploited for translational regulation by genes that use upstream open reading frames (uORFs) to control how often ribosomes reach the main coding sequence.

Question 2

A mutation destroys the Shine-Dalgarno sequence upstream of a bacterial gene while leaving the AUG start codon and downstream coding sequence intact. What is the most likely consequence?

• A. The ribosome will initiate at the next downstream AUG, producing a slightly truncated protein
• B. Translation efficiency will drop dramatically because the 30S subunit cannot position itself at the correct start codon
• C. The ribosome will switch to the eukaryotic scanning mechanism, using the 5' end of the mRNA instead
• D. The reading frame will shift, producing a frameshifted protein from the same AUG

Answer: (B)

In prokaryotes, start codon selection depends on direct base-pairing between the Shine-Dalgarno sequence and the 16S rRNA in the 30S subunit. This interaction physically positions the 30S subunit so the AUG falls precisely in the P site. Without a Shine-Dalgarno sequence, the 30S subunit cannot find and position itself at the correct AUG — translation drops dramatically or is eliminated. There is no fallback scanning mechanism in prokaryotes, and the mRNA may contain many internal AUG triplets that are not the correct initiation site. The Shine-Dalgarno/AUG pair is the primary determinant of translation efficiency per gene in bacteria.

Question 3

In prokaryotes, a single mRNA molecule can encode and be translated into multiple separate proteins, each initiated independently.

• T. True
• F. False

Answer: True

Prokaryotic mRNAs are often polycistronic — encoding multiple proteins on a single transcript. This is possible because each coding sequence has its own independent Shine-Dalgarno sequence positioned upstream of its own AUG. The 30S subunit can independently find and initiate at each Shine-Dalgarno/AUG pair, producing separate proteins from a single mRNA. This is fundamentally impossible in eukaryotes using the cap-dependent scanning model, which can only recognize one 5' end — eukaryotic mRNAs are almost always monocistronic as a result.

Question 4

If the ribosome initiates translation at the wrong AUG, primarily the first few amino acids of the resulting protein are affected; the rest of the sequence is read correctly because the genetic code is the same downstream.

• T. True
• F. False

Answer: False

The start codon does not just specify the first amino acid — it establishes the reading frame for the entire downstream sequence. Codons are read as non-overlapping triplets starting from the initiation site. If an incorrect AUG is used, every subsequent triplet is read in the wrong phase, producing a completely different amino acid sequence until a premature stop codon is encountered in the wrong frame. The result is almost always a nonfunctional protein and typically rapid mRNA degradation via nonsense-mediated decay. This is why translation initiation accuracy is so critical and why eukaryotes invest in numerous initiation factors.

Question 5

Why do eukaryotes require at least twelve initiation factors (eIFs) while prokaryotes manage with only three (IFs), and what problem does this greater complexity solve?

Model answer: The complexity reflects the fundamentally different initiation strategy. Prokaryotes use a simple mechanism: Shine-Dalgarno base-pairing positions the 30S subunit directly at the correct AUG, requiring only factors to handle fMet-tRNA delivery (IF2), prevent premature 50S joining (IF3), and block the A site (IF1). Eukaryotes cannot use this approach because their mRNAs lack Shine-Dalgarno sequences and are usually monocistronic. Instead, the 40S subunit must recognize the 5' cap (requiring eIF4E), be loaded onto the mRNA (eIF4G as scaffold), unwind 5' UTR secondary structure during scanning (eIF4A helicase), carry the initiator Met-tRNA (eIF2), scan to the first AUG, decode Kozak context, and coordinate 60S subunit joining (eIF5, eIF5B). Each step is a distinct molecular event requiring dedicated factors. The additional complexity also enables elaborate regulatory control: phosphorylating eIF2α during cellular stress globally suppresses translation initiation, a regulatory mechanism impossible with the simple SD-based system.