title: Dual effect of chloramphenicol peptides on ribosome inhibition.
abstract: Chloramphenicol peptides were recently established as useful tools for probing nascent polypeptide chain interaction with the ribosome, either biochemically, or structurally. Here, we present a new 10mer chloramphenicol peptide, which exerts a dual inhibition effect on the ribosome function affecting two distinct areas of the ribosome, namely the peptidyl transferase center and the polypeptide exit tunnel. According to our data, the chloramphenicol peptide bound on the chloramphenicol binding site inhibits the formation of both acetyl-phenylalanine-puromycin and acetyl-lysine-puromycin, showing, however, a decreased peptidyl transferase inhibition compared to chloramphenicol-mediated inhibition per se. Additionally, we found that the same compound is a strong inhibitor of green fluorescent protein synthesis in a coupled in vitro transcription-translation assay as well as a potent inhibitor of lysine polymerization in a poly(A)-programmed ribosome, showing that an additional inhibitory effect may exist. Since chemical protection data supported the interaction of the antibiotic with bases A2058 and A2059 near the entrance of the tunnel, we concluded that the extra inhibition effect on the synthesis of longer peptides is coming from interactions of the peptide moiety of the drug with residues comprising the ribosomal tunnel, and by filling up the tunnel and blocking nascent chain progression through the restricted tunnel. Therefore, the dual interaction of the chloramphenicol peptide with the ribosome increases its inhibitory effect and opens a new window for improving the antimicrobial potency of classical antibiotics or designing new ones.
authors.affiliation: Department of Biochemistry, School of Medicine, University of Patras, 26500, Patras, Greece.
authors.forename: Anthony
authors.identifier: 0000-0003-3914-7408
authors.initials: A
authors.lastname: Bougas
authors.affiliation: Department of Biochemistry, School of Medicine, University of Patras, 26500, Patras, Greece.
authors.forename: Ioannis A
authors.initials: IA
authors.lastname: Vlachogiannis
authors.affiliation: Department of Chemistry, University of Patras, Patras, Greece.
authors.forename: Dimitrios
authors.initials: D
authors.lastname: Gatos
authors.affiliation: Gene Center and Department of Biochemistry, Ludwig-Maximilians-University of Munich, Feodor- Lynen-Strasse 25, 81377, Munich, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Arenz
authors.affiliation: Department of Biochemistry, School of Medicine, University of Patras, 26500, Patras, Greece. dinosg@upatras.gr.
authors.forename: George P
authors.initials: GP
authors.lastname: Dinos
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D002701:Chloramphenicol / Q000031:analogs & derivatives / Q000138:chemical synthesis / Q000494:pharmacology*; D048168:Escherichia coli K12 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005449:Fluorenes / Q000737:chemistry*; D049452:Green Fluorescent Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics; D000067556:Internal Ribosome Entry Sites / Q000187:drug effects; D008958:Models, Molecular; D010455:Peptides / Q000138:chemical synthesis / Q000494:pharmacology*; D010458:Peptidyl Transferases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D011061:Poly A / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D014176:Protein Biosynthesis / Q000187:drug effects*; D011500:Protein Synthesis Inhibitors / Q000138:chemical synthesis / Q000494:pharmacology*; D011691:Puromycin / Q000494:pharmacology; D012270:Ribosomes / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism
chemical_list: D005449:Fluorenes; D000067556:Internal Ribosome Entry Sites; D010455:Peptides; D011500:Protein Synthesis Inhibitors; D049452:Green Fluorescent Proteins; D011061:Poly A; D011691:Puromycin; D002701:Chloramphenicol; D010458:Peptidyl Transferases
keywords: Antibiotics; Chloramphenicol-derivatives; Nascent peptidyl-tRNA mimics; Peptidyl-tRNA analogs; Ribosomal tunnel