title: Negamycin induces translational stalling and miscoding by binding to the small subunit head domain of the Escherichia coli ribosome.
abstract: Negamycin is a natural product with broad-spectrum antibacterial activity and efficacy in animal models of infection. Although its precise mechanism of action has yet to be delineated, negamycin inhibits cellular protein synthesis and causes cell death. Here, we show that single point mutations within 16S rRNA that confer resistance to negamycin are in close proximity of the tetracycline binding site within helix 34 of the small subunit head domain. As expected from its direct interaction with this region of the ribosome, negamycin was shown to displace tetracycline. However, in contrast to tetracycline-class antibiotics, which serve to prevent cognate tRNA from entering the translating ribosome, single-molecule fluorescence resonance energy transfer investigations revealed that negamycin specifically stabilizes near-cognate ternary complexes within the A site during the normally transient initial selection process to promote miscoding. The crystal structure of the 70S ribosome in complex with negamycin, determined at 3.1 Å resolution, sheds light on this finding by showing that negamycin occupies a site that partially overlaps that of tetracycline-class antibiotics. Collectively, these data suggest that the small subunit head domain contributes to the decoding mechanism and that small-molecule binding to this domain may either prevent or promote tRNA entry by altering the initial selection mechanism after codon recognition and before GTPase activation.
authors.affiliation: Discovery Sciences, AstraZeneca R&D Boston, Waltham, MA 02451;
authors.forename: Nelson B
authors.initials: NB
authors.lastname: Olivier
authors.affiliation: Department of Physiology and Biophysics & the Tri-Institutional PhD Program in Chemical Biology, Weill Cornell Medical College, New York, NY 10065;
authors.forename: Roger B
authors.initials: RB
authors.lastname: Altman
authors.affiliation: Department of Molecular Cell Biology, California Institute of Quantitative Biosciences, University of California, Berkeley, CA 94720; and.
authors.forename: Jonas
authors.initials: J
authors.lastname: Noeske
authors.affiliation: Departments of Chemistry and.
authors.forename: Gregory S
authors.initials: GS
authors.lastname: Basarab
authors.affiliation: Discovery Sciences, AstraZeneca R&D Boston, Waltham, MA 02451;
authors.forename: Erin
authors.initials: E
authors.lastname: Code
authors.affiliation: Discovery Sciences, AstraZeneca R&D Boston, Waltham, MA 02451;
authors.forename: Andrew D
authors.initials: AD
authors.lastname: Ferguson
authors.affiliation: Discovery Sciences, AstraZeneca R&D Boston, Waltham, MA 02451;
authors.forename: Ning
authors.initials: N
authors.lastname: Gao
authors.affiliation: Discovery Sciences, AstraZeneca R&D Boston, Waltham, MA 02451;
authors.forename: Jian
authors.initials: J
authors.lastname: Huang
authors.affiliation: Department of Physiology and Biophysics & the Tri-Institutional PhD Program in Chemical Biology, Weill Cornell Medical College, New York, NY 10065;
authors.forename: Manuel F
authors.initials: MF
authors.lastname: Juette
authors.affiliation: Discovery Sciences, AstraZeneca R&D Boston, Waltham, MA 02451;
authors.forename: Stephania
authors.initials: S
authors.lastname: Livchak
authors.affiliation: Departments of Chemistry and.
authors.forename: Matthew D
authors.initials: MD
authors.lastname: Miller
authors.affiliation: Discovery Sciences, AstraZeneca R&D Boston, Waltham, MA 02451;
authors.forename: D Bryan
authors.initials: DB
authors.lastname: Prince
authors.affiliation: Department of Molecular Cell Biology, California Institute of Quantitative Biosciences, University of California, Berkeley, CA 94720; and.
authors.forename: Jamie H D
authors.initials: JH
authors.lastname: Cate
authors.affiliation: Biosciences, Infection Innovative Medicines Unit, AstraZeneca R&D Boston, Waltham, MA 02451 scb2005@med.cornell.edu Ed.Buurman@astrazeneca.com.
authors.forename: Ed T
authors.initials: ET
authors.lastname: Buurman
authors.affiliation: Department of Physiology and Biophysics & the Tri-Institutional PhD Program in Chemical Biology, Weill Cornell Medical College, New York, NY 10065; scb2005@med.cornell.edu Ed.Buurman@astrazeneca.com.
authors.forename: Scott C
authors.initials: SC
authors.lastname: Blanchard
mesh_terms: D000599:Amino Acids, Diamino / Q000494:pharmacology; D000900:Anti-Bacterial Agents / Q000378:metabolism / Q000494:pharmacology*; D020029:Base Pairing; D001665:Binding Sites; D001667:Binding, Competitive; D018360:Crystallography, X-Ray; D024901:Drug Resistance, Multiple, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000187:drug effects* / Q000235:genetics; D008911:Minocycline / Q000031:analogs & derivatives / Q000494:pharmacology; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D017354:Point Mutation; D014176:Protein Biosynthesis / Q000187:drug effects; D012329:RNA, Bacterial / Q000737:chemistry / Q000187:drug effects* / Q000235:genetics / Q000502:physiology; D012336:RNA, Ribosomal, 16S / Q000737:chemistry / Q000187:drug effects* / Q000235:genetics / Q000502:physiology; D012343:RNA, Transfer / Q000378:metabolism; D012270:Ribosomes / Q000187:drug effects* / Q000648:ultrastructure; D013753:Tetracycline Resistance / Q000235:genetics; D013754:Tetracyclines / Q000378:metabolism / Q000494:pharmacology; D000078304:Tigecycline
chemical_list: D000599:Amino Acids, Diamino; D000900:Anti-Bacterial Agents; D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S; D013754:Tetracyclines; D000078304:Tigecycline; D012343:RNA, Transfer; C100200:negamycin; D008911:Minocycline
keywords: antibiotic; fidelity; negamycin; ribosome; translation