Tetracenomycin X sequesters peptidyl-tRNA during translation of QK motifs

With antibiotic-resistant bacteria threatening our ability to treat common infections, new lead compounds with distinct target binding sites and limited cross-resistance are urgently needed. Natural products that inhibit the bacterial ribosome - a target for more than half of the antibiotics in use today - are a promising source of such leads and have the potential to be developed into potent drugs through structure-guided design. However, because the mechanisms of action of many of these compounds are not well understood, they are often poor candidates for a structure-based approach. Here, we use inverse toeprinting coupled to next-generation sequencing to show that the aromatic polyketide tetracenomycin X (TcmX) primarily inhibits the formation of a peptide bond between an incoming aminoacyl-tRNA and a terminal Gln-Lys (QK) motif in the nascent polypeptide. Using cryogenic electron microscopy, we reveal that translation inhibition at QK motifs occurs via an unusual mechanism involving sequestration of the 3 adenosine of peptidyl-tRNALys in the drug-occupied nascent polypeptide exit tunnel of the ribosome. Our study provides mechanistic insights into the mode of action of TcmX on the bacterial ribosome and suggests a path forward for the development of novel antibiotics based on a common aromatic polyketide scaffold.