ИСТИНА

Many antibiotics that target the eukaryotic ribosome represent potential antitumor agents. For some of them, their molecular interactions have been extensively studied in structural terms while functional details of their activity remain incompletely understood. Using toe-printing technique, we analyzed effects of several translation elongation inhibitors in mammalian cell extract. When pre-incubated with the lysate before mRNA addition, both aminoglycoside antibiotics and translocation inhibitors stopped elongating ribosome at the very beginning of the coding region. In contrast, several antibiotics that affected the peptidyl transferase center (PTC) of the 60S subunit demonstrated complex patterns of toe-print signals along the mRNA. We investigated this phenomenon in detail using a model of antitumor drug harringtonine, which had been used recently in ribosome profiling studies. We showed that harringtonine and its close derivative homoharringtonine specifically halted elongating ribosomes at Lys, Arg or Tyr codons positioned in the P-site producing strong toe-print bands in the corresponding positions. Bioinformatic analysis of ribosome profiling data generally confirmed this conclusion in a transcriptome-wide scale. Molecular modeling based on recently resolved structures of eukaryotic ribosome let us to assume that in addition to the spatial interference of homoharringtonine in the A-site it may also require the interaction of the antibiotic with the peptidyl group of the P-site bound tRNA. Finally, we unexpectedly revealed the same pattern of elongation arrest for a chemically distinct trichothecene antibiotic, T-2 toxin, while another PTC inhibitor of this group, diacetoxyscirpenol, produced a completely distinct pattern of ribosome stops. Our data suggests that the mechanism of inhibition of the protein synthesis by PTC inhibitors is more complex than it was originally proposed.