For the last few decades, the number of multidrug resistant pathogens has increased steadily whereas the number of new antibiotics entering the market has decreased dramatically, highlighting the urgent need to discover new antimicrobials. We report that the recently discovered antibiotic

tetracenomycin X (TcmX) allows translation of most oligopeptides and, similarly to macrolide antibiotics, only inhibits translation of specific nascent peptides containing blocking motifs. TcmX binds within the nascent polypeptide exit tunnel of the ribosome, adjacent to the binding site of macrolide

antibiotics like erythromycin. Despite the mechanistic similarities with macrolides, we show that TcmX inhibits peptide bond formation in the PTC through a novel context-dependent mechanism, whereby nascent peptides containing a Gln-Lys (QK) motif are retained within the exit tunnel and trapping the 3'

adenosine of the P-site lysyl-tRNA towards the tunnel-bound drug. Our study provides mechanistic insights into the mode of action of TcmX on the prokaryotic ribosome, paving the way for the development of novel antimicrobial molecules based on a common aromatic polyketide scaffold.