Questions: Translation Elongation and Elongation Factors

Question 1

A mutation eliminates EF-Tu's GTPase activity so it can bind GTP but cannot hydrolyze it. What is the primary consequence for translation?

• A. The ribosome cannot translocate, because EF-Tu normally drives mRNA movement after peptide bond formation
• B. Peptide bond formation is blocked, because GTP hydrolysis provides the energy needed to form the amide bond
• C. Incorrectly matched aminoacyl-tRNAs cannot be efficiently rejected, leading to increased mistranslation and amino acid misincorporation
• D. EF-Ts cannot recharge EF-Tu with GTP, depleting the pool of active elongation factor over time

Answer: (C)

GTP hydrolysis by EF-Tu is a proofreading mechanism, not the energy source for peptide bond formation (which comes from the high-energy aminoacyl-tRNA ester bond). When an aminoacyl-tRNA arrives with the correct anticodon, the codon-anticodon geometry stimulates GTPase activity, releasing EF-Tu and allowing full accommodation. Without GTP hydrolysis, EF-Tu cannot release — but more importantly, the kinetic delay that allows incorrect tRNAs to dissociate is eliminated. Incorrect tRNAs would be accommodated at higher rates, dramatically increasing errors. Option D (EF-Ts) is wrong because EF-Ts exchanges GDP for GTP on EF-Tu — if EF-Tu never hydrolyzes GTP, GDP doesn't accumulate.

Question 2

Peptide bond formation during elongation is catalyzed by:

• A. EF-G, which positions the P-site tRNA correctly for nucleophilic attack by the A-site amino acid
• B. A dedicated peptidyl transferase protein embedded in the large ribosomal subunit
• C. The 23S (prokaryote) or 28S (eukaryote) ribosomal RNA — the ribosome functions as a ribozyme
• D. EF-Tu, following GTP hydrolysis and release from the aminoacyl-tRNA

Answer: (C)

The discovery that peptidyl transferase activity resides in the ribosomal RNA, not a protein, established the ribosome as a ribozyme — one of the most important findings in molecular biology. The 23S rRNA (prokaryotes) or 28S rRNA (eukaryotes) positions the P-site peptidyl-tRNA and A-site aminoacyl-tRNA for nucleophilic attack, catalyzing peptide bond formation. Ribosomal proteins stabilize rRNA structure but are not the catalytic center. EF-G drives translocation (step 3), not peptide bond formation.

Question 3

GTP hydrolysis by EF-Tu provides the energy that forms the peptide bond between successive amino acids during elongation.

• T. True
• F. False

Answer: False

This is a common misconception. GTP hydrolysis by EF-Tu functions as a proofreading verification step — it introduces a kinetic delay that allows incorrectly matched tRNAs to dissociate before they are permanently accommodated. The energy for peptide bond formation comes from the high-energy ester bond linking the amino acid to the 3' end of the tRNA. When the peptide is transferred from P-site tRNA to A-site tRNA, that ester bond is broken, releasing energy that drives the thermodynamically unfavorable peptide bond forward.

Question 4

Elongation factors EF-Tu and EF-G transiently associate with the ribosome during each elongation cycle — they bind, perform their function, and dissociate, rather than remaining as permanent ribosomal components.

• T. True
• F. False

Answer: True

Both EF-Tu and EF-G are cycling factors. EF-Tu·GTP delivers the aminoacyl-tRNA, GTP is hydrolyzed, and EF-Tu·GDP is released and then recharged by EF-Ts. EF-G·GTP then binds to drive translocation, GTP is hydrolyzed, and EF-G·GDP is released. This cycling behavior means each factor participates in every elongation cycle but spends most of its time free in solution. The misconception that they are permanent ribosome components would imply the ribosome is much larger than it is and that each factor can only serve one ribosome.

Question 5

Explain the role of GTP hydrolysis in EF-Tu's proofreading mechanism, and how it improves the fidelity of amino acid incorporation.

Model answer: When EF-Tu·GTP delivers an aminoacyl-tRNA to the A site, GTP hydrolysis does not occur immediately. The ribosome first checks whether the anticodon matches the mRNA codon. A correct codon-anticodon interaction induces a conformational change that stimulates EF-Tu's GTPase activity. This hydrolysis step introduces a kinetic pause between initial recognition and full accommodation of the tRNA into the A site. Incorrectly matched tRNAs trigger GTP hydrolysis much more slowly — giving them time to dissociate before the irreversible peptide bond is formed. By coupling commitment to an upstream verification step, the ribosome achieves error rates of ~1 per 10,000 amino acids despite operating at 15–20 residues per second.

The mechanism is called 'kinetic proofreading' and relies on the temporal separation of two steps: initial selection (codon-anticodon matching) and accommodation (full A-site binding). GTP hydrolysis creates an irreversible transition that incorrect tRNAs rarely survive. The energy cost of GTP is the price of accuracy — without it, the ribosome would be faster but far less faithful, producing catastrophically misfolded proteins.