| Puromycin is an aminonucleoside antibiotic produced by Streptomyces alboniger and structurally mimics the 3' end of an aminoacylated tyrosyl-tRNA, with a modified adenosine base covalently linked to a tyrosine-like amino acid moiety; the critical structural distinction from genuine aminoacyl-tRNA is the bond connecting the amino acid to the nucleoside, a stable peptide bond in puromycin rather than the labile ester bond found in charged tRNA. During translation elongation, puromycin enters the ribosomal A-site in place of the incoming aminoacyl-tRNA, where its free amino group accepts the nascent polypeptide chain from the P-site peptidyl-tRNA through the catalytic activity of the ribosomal peptidyl transferase center, exactly as a genuine aminoacyl-tRNA would during normal peptide bond formation. Because the resulting peptide bond linking the nascent chain to puromycin cannot be cleaved by any subsequently arriving aminoacyl-tRNA, this incorporation event is irreversible: no further chain extension can occur, and the ribosome dissociates, releasing the truncated nascent chain now carrying puromycin covalently attached at its C-terminus as a peptidyl-puromycin adduct. This mechanism operates as a competitive, concentration-dependent process, since puromycin at high concentrations competes broadly with aminoacyl-tRNA for A-site occupancy throughout elongation, producing widespread premature chain termination and general inhibition of protein synthesis, whereas at sufficiently low concentrations puromycin only inefficiently competes with the standard elongation cycle and instead tends to react preferentially near translational pause points such as stop codons, allowing selective labeling of near-full-length or full-length protein rather than indiscriminate truncation. The resulting peptidyl-puromycin adducts generated during premature termination are recognized as aberrant translation products and are rapidly cleared through the proteasome in a dose- and time-dependent manner, and at low puromycin concentrations, the flux of these prematurely terminated polypeptides into proteasomal degradation increases recovery of peptide-MHC class I complexes at the cell surface, directly linking puromycin-induced translational drop-off products to the antigen presentation pathway; at sustained higher puromycin exposure, however, MHC class I surface export becomes inhibited as polyubiquitinated protein accumulates, indicating that the proteolytic and trafficking machinery handling these adducts becomes saturated. |
