Poster abstracts

Poster number 91 submitted by James Marks

Context Specific Action of Linezolid and Chloramphenicol

James Marks (Center for Biomolecular Sciences, University of Illinois at Chicago), Amira Kefi (Center for Biomolecular Sciences, University of Illinois at Chicago), Dorota Klepacki (Center for Biomolecular Sciences, University of Illinois at Chicago), Krishna Kannan (Center for Biomolecular Sciences, University of Illinois at Chicago), Nora Vazquez-Laslop (Center for Biomolecular Sciences, University of Illinois at Chicago), Alexander Mankin (Center for Biomolecular Sciences, University of Illinois at Chicago)

Abstract:
The generally accepted mechanism of action of the classic antibiotic chloramphenicol (CHL) and the newer drug linezolid (LNZ) is inhibition of peptide bond formation. Because both antibiotics bind in the A-site of the peptidyl transferase center (PTC), they obstruct accommodation of aminoacyl-tRNAs and, thus, it is assumed that they prevent the formation of any peptide bond. Thereby, it is anticipated that exposure of bacterial cells to CHL or LZD should non-specifically and equally efficiently arrest translating ribosomes at any codon along mRNA.
We employed the genome-wide technique of ribosome profiling to examine drug-induced changes in the distribution of ribosomes along cellular mRNAs. Neither of the drugs produced the expected uniform arrest of ribosomes on mRNA. Strikingly, the data revealed that the antibiotic-arrested ribosomes preferentially carried a nascent peptide whose penultimate amino acids were Ala, and less frequently Ser or Thr. Subsequent biochemical experiments in a cell-free translation system confirmed that neither one of these compounds has a global inhibitory effect but instead, block formation of only specific peptide bonds, based on the sequence context of the protein being made.
Remarkably, the CHL mediated programmed translation arrest which regulates inducible drug resistance in bacteria, exploits the same principle: the arresting sequence of the regulatory nascent peptides contains an Ala or Thr residues in their penultimate positions.
Our findings suggest that the mechanisms of action of this important class of antibiotics have been largely misunderstood and reveal that the nascent peptide, in a sequence specific manner, affects interactions of the substrates with the ribosomal A site and define the preferential sites of action of the PTC inhibitor by either promoting the antibiotic binding or slowing the rate of accommodation of aminoacyl-tRNAs.

References:
Oh E, et al. (2011) Selective ribosome profiling reveals the cotranslational chaperone action of trigger factor in vivo. Cell 147(6):1295-1308.

Lovett PS (1996) Translation attenuation regulation of chloramphenicol resistance in bacteria - A review. Gene 179:157-162.

O'Shea JP, et al. (2013) pLogo: a probabilistic approach to visualizing sequence motifs. Nat Methods 10(12):1211-1212.

Leach KL, et al. (2007) The site of action of oxazolidinone antibiotics in living bacteria and in human mitochondria. Mol Cell 26(3):393-402.

Dunkle JA, Xiong L, Mankin AS, Cate JH (2010) Structures of the Escherichia coli ribosome with antibiotics bound near the peptidyl transferase center explain spectra of drug action. Proc Natl Acad Sci USA 107(40):17152-17157.

Keywords: Antibiotic, Ribosome, Translation