Which simple biochemical signature in a cell-free translation system would best distinguish a peptidyl transferase inhibitor (like chloramphenicol) from an inhibitor of tRNA binding (like tetracycline)?

• A. Observation of polysome collapse under electron microscopy unequivocally tells if a drug is a tRNA binder or peptidyl transferase inhibitor because polysome shapes are drug-specific and easily interpreted without additional assays.
• B. Only transcriptional profiling can distinguish these mechanisms because translation assays are incapable of resolving whether A-site binding or peptidyl transfer is inhibited.
• C. A universal decrease in cell-free ATP levels would uniquely identify peptidyl transferase inhibitors, while increased ATP would identify tRNA binding inhibitors.
• D. Accumulation of short peptidyl-tRNA species on SDS-PAGE and stalled ribosome-associated peptides indicates peptidyl transferase inhibition, whereas near absence of nascent peptides and lack of A-site toeprints indicates tRNA binding inhibition.

Answer: (D)