Chloramphenicol and Linezolid Selectively Trap A-Site tRNAs in the Ribosome

Abstract The ribosome makes all the proteins in the cell by adding amino acids to the protein chain one by one. The amino acids are delivered to the so-called A site of the functional center of the ribosome by special molecules, the tRNAs. Because of the ribosome’s essential role in cellular life, it is not surprising that numerous antibiotics inhibit bacterial growth by disrupting the function of the ribosome. For example, linezolid and chloramphenicol, important antibiotics in the clinic, bind to the ribosomal A site and, therefore, were thought to completely inhibit protein synthesis by blocking the entry of any amino acid into the ribosome. However, there were numerous in vitro experiments reported in the literature showing that ribosomes, while associated with linezolid or chloramphenicol, were still able to form small protein chains. Hence, we hypothesized that neither of these antibiotics were universal inhibitors of protein synthesis. The goal of my Ph.D. work was to understand the mode of action of linezolid and chloramphenicol. By utilizing genome-wide approaches in living bacterial cells, as well as biochemistry, bioinformatics, and single molecule Förster resonance energy transfer we learned that chloramphenicol and linezolid: 1- Arrest the ribosome depending on the amino acid sequence being made. Specifically, these antibiotics block delivery of the amino acid into the A site only when the penultimate amino acid of the nascent protein is an alanine, a threonine or a serine. However, this rule is broken if the delivered amino acid is a glycine. 2- Trap the ribosome in a functionally inactive state. The tRNAs can deliver the proper amino acid to the A site but the antibiotics incapacitate the ribosome from incorporate it into the nascent protein. Eventually, the amino acid-tRNA pair dissociates from the ribosome and new, but still unreactive, amino acid delivery attempts occur. This work revealed that, contrary to what was proposed in the past, chloramphenicol and linezolid are selective, context specific, inhibitors of protein synthesis. Understanding the mechanistic details of antibiotic action should contribute to the rational design of novel antibacterials and more efficient treatments to combat infections.