Questions: Translation Elongation and Termination: Peptide Bond Formation

Question 1

A drug blocks peptide bond formation during translation without affecting tRNA delivery, GTP hydrolysis, or translocation. What is the drug's most likely target?

• A. EF-Tu, the elongation factor that delivers aminoacyl-tRNAs to the A site
• B. The peptidyl transferase center — specifically the 23S (or 28S) rRNA that catalyzes the reaction
• C. EF-G, the translocase that moves tRNAs and mRNA by one codon
• D. A ribosomal protein enzyme in the large subunit that stabilizes the transition state

Answer: (B)

Peptide bond formation is catalyzed by the peptidyl transferase center of the large ribosomal subunit, and this activity resides in the 23S rRNA (prokaryotes) or 28S rRNA (eukaryotes) — making the ribosome a ribozyme. There is no dedicated protein enzyme for this step. A drug targeting peptide bond formation without affecting delivery or translocation would have to act on this RNA-based catalytic center. Option D is the most tempting wrong answer — many students assume there must be a protein enzyme.

Question 2

What is the function of GTP hydrolysis by EF-Tu during aminoacyl-tRNA delivery, and why does it improve accuracy beyond what base pairing alone achieves?

• A. GTP hydrolysis provides energy to force the aminoacyl-tRNA into the A site against electrostatic repulsion
• B. GTP hydrolysis triggers a conformational change that releases EF-Tu only after correct codon-anticodon pairing is verified, allowing incorrect tRNAs to dissociate before accommodation — kinetic proofreading
• C. GTP hydrolysis powers translocation of the ribosome by one codon after delivery is complete
• D. GTP hydrolysis activates the aminoacyl-tRNA by phosphorylating the amino acid before peptide bond formation

Answer: (B)

This is kinetic proofreading. EF-Tu holds the charged tRNA in a configuration that cannot participate in peptide bond formation. Correct codon-anticodon pairing triggers a conformational change in the ribosome that stimulates GTP hydrolysis by EF-Tu, releasing the factor and allowing the tRNA to fully accommodate in the A site. Incorrect tRNAs lack the geometric fit needed to trigger this change and dissociate before GTP hydrolysis — a second selection step on top of base pairing. This two-stage discrimination achieves ~1 error per 10,000 amino acids, far better than the ~1 in 100 that base pairing alone would produce.

Question 3

When a stop codon enters the A site of the ribosome, termination is triggered by a release factor that structurally mimics a tRNA.

• T. True
• F. False

Answer: True

Release factors (RF1/RF2 in prokaryotes, eRF1 in eukaryotes) have an overall shape that resembles a tRNA, allowing them to fit into the A site. This molecular mimicry positions the factor's catalytic domain near the peptidyl transferase center, where it triggers hydrolysis of the ester bond connecting the completed polypeptide to the final tRNA — releasing the finished protein. This is an elegant example of structural mimicry: a protein solution to a problem first 'solved' by RNA.

Question 4

Peptide bond formation in the ribosome is catalyzed by a protein enzyme called peptidyl transferase, which is encoded by a ribosomal protein gene.

• T. True
• F. False

Answer: False

Peptide bond formation is catalyzed by the ribosomal RNA itself — specifically the 23S rRNA in prokaryotes and 28S rRNA in eukaryotes — making the ribosome a ribozyme. There is no protein enzyme responsible for this reaction. This was a major discovery confirming the RNA World hypothesis: the most fundamental step in protein synthesis is catalyzed by RNA, not protein. Ribosomal proteins play structural and regulatory roles but are not the catalytic component for peptide bond formation.

Question 5

Explain how kinetic proofreading by EF-Tu achieves an error rate far lower than codon-anticodon base pairing alone could provide.

Model answer: Codon-anticodon base pairing provides one level of discrimination between correct and incorrect tRNAs, but the free energy difference between correct (Watson-Crick) and near-cognate base pairs is not large enough to achieve 1 error in 10,000 by thermodynamics alone. EF-Tu adds a second, kinetically independent discrimination step: it holds the aminoacyl-tRNA in a pre-accommodation state where peptide bond formation cannot occur. Only correct codon-anticodon pairing triggers a ribosomal conformational change that stimulates GTP hydrolysis by EF-Tu, releasing the factor and allowing tRNA accommodation. Incorrect tRNAs dissociate before this conformational change — they have had two independent opportunities to be rejected. The product of two sequential error rates (each ~1 in 100) gives ~1 in 10,000.

Kinetic proofreading is a general principle that appears wherever cells need accuracy beyond what equilibrium chemistry provides: it inserts irreversible steps (GTP hydrolysis) that allow incorrect intermediates to be discarded before the reaction is committed, at the cost of energy expenditure.