You are given an unknown antibiotic and want to determine which of four mechanisms it uses: (A) blocks aminoacyl-tRNA binding to the A site, (B) causes mistranslation by altering decoding, (C) blocks peptide bond formation at the peptidyl transferase center, or (D) blocks the polypeptide exit tunnel. Which single experimental approach will most directly discriminate among all four mechanisms, and what are the expected diagnostic results for each mechanism?

Perform an in vitro translation assay combining (1) radiolabeled amino acid incorporation to assess total peptide synthesis, (2) toeprinting to measure tRNA/mRNA positioning at the A site versus stalled complexes, and (3) SDS-PAGE autoradiography to detect truncated versus full-length stalled nascent chains: A gives minimal radiolabel incorporation with lack of toeprint signals for A-site occupancy; B gives normal incorporation but heterogeneous peptide masses and misincorporation detected by mass spectrometry; C gives accumulation of single amino-acid–linked peptidyl-tRNAs with short peptides on gel; D gives full-length, peptidyl-tRNA–associated stalled complexes that are long but trapped and localized to the ribosome, visible as high-molecular-weight bands and strong toeprint at the stall site.

Use a simple Gram-stain followed by MIC determination: Gram staining reveals whether the bacteria are Gram-negative or Gram-positive, and MIC shifts indicate which of the four mechanisms is present, because each mechanism has a characteristic MIC range specific to Gram type.

Run an SDS-PAGE of total cellular proteins and observe band intensity: disappearance of all bands means tRNA binding inhibition, smearing indicates mistranslation, novel low-molecular-weight bands indicate peptide bond inhibition, and presence of intact high-molecular-weight complexes indicates exit tunnel blockage.

Measure bacterial ATP levels in culture after drug treatment: complete ATP depletion corresponds to ribosomal exit tunnel blockage, moderate ATP drop indicates peptide bond inhibition, small changes reflect tRNA binding interference, and random spikes imply mistranslation.