title: Mechanism of Tet(O)-mediated tetracycline resistance.
abstract: Tet(O) is an elongation factor-like protein which confers resistance to the protein synthesis inhibitor tetracycline by promoting the release of the drug from its inhibitory site on the ribosome. Here we investigated the interaction of Tet(O) with the elongating ribosome and show, using dimethyl sulfate (DMS) probing and binding assays, that it interacts preferentially with the post-translocational ribosome. Furthermore, using an XTP-dependent mutant of Tet(O), we demonstrated that Tet(O) induces conformational rearrangements within the ribosome which can be detected by EF-Tu, and manifested as a stimulation in the GTPase activity of this elongation factor. As such, these conformational changes probably involve the ribosomal GTPase-associated center and, accordingly, Tet(O) alters the DMS modification pattern of the L11 region. Additionally, tetracycline binding is associated with an E(a) of 58 kJ/mol. These results suggest a model where both Tet(O) and tetracycline induce a conformational change in functionally opposite directions and the Tet(O)-induced conformation persists after it has left the ribosome; this prevents rebinding of the drug while allowing productive A-site occupation by a ternary complex in the presence of tetracycline.
authors.affiliation: Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, AB, T6G 2H7, Canada.
authors.forename: Sean R
authors.initials: SR
authors.lastname: Connell
authors.forename: Catharine A
authors.initials: CA
authors.lastname: Trieber
authors.forename: George P
authors.initials: GP
authors.lastname: Dinos
authors.forename: Edda
authors.initials: E
authors.lastname: Einfeldt
authors.forename: Diane E
authors.initials: DE
authors.lastname: Taylor
authors.forename: Knud H
authors.initials: KH
authors.lastname: Nierhaus
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism*; D016123:Campylobacter jejuni / Q000187:drug effects / Q000502:physiology; D002352:Carrier Proteins; D024881:Drug Resistance, Bacterial / Q000235:genetics* / Q000502:physiology; D020652:Peptide Elongation Factor 2 / Q000378:metabolism; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology*; D012270:Ribosomes / Q000378:metabolism; D013696:Temperature; D013752:Tetracycline / Q000494:pharmacology*
chemical_list: D001426:Bacterial Proteins; D002352:Carrier Proteins; D020652:Peptide Elongation Factor 2; D011500:Protein Synthesis Inhibitors; C083139:Tet O resistance protein, Bacteria; D013752:Tetracycline