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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.
AuthorsNelson B Olivier, Roger B Altman, Jonas Noeske, Gregory S Basarab, Erin Code, Andrew D Ferguson, Ning Gao, Jian Huang, Manuel F Juette, Stephania Livchak, Matthew D Miller, D Bryan Prince, Jamie H D Cate, Ed T Buurman, Scott C Blanchard
JournalProceedings of the National Academy of Sciences of the United States of America (Proc Natl Acad Sci U S A) Vol. 111 Issue 46 Pg. 16274-9 (Nov 18 2014) ISSN: 1091-6490 [Electronic] United States
PMID25368144 (Publication Type: Journal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't)
Chemical References
  • Amino Acids, Diamino
  • Anti-Bacterial Agents
  • RNA, Bacterial
  • RNA, Ribosomal, 16S
  • Tetracyclines
  • Tigecycline
  • RNA, Transfer
  • negamycin
  • Minocycline
Topics
  • Amino Acids, Diamino (pharmacology)
  • Anti-Bacterial Agents (metabolism, pharmacology)
  • Base Pairing
  • Binding Sites
  • Binding, Competitive
  • Crystallography, X-Ray
  • Drug Resistance, Multiple, Bacterial (genetics)
  • Escherichia coli (drug effects, genetics)
  • Minocycline (analogs & derivatives, pharmacology)
  • Models, Molecular
  • Nucleic Acid Conformation
  • Point Mutation
  • Protein Biosynthesis (drug effects)
  • RNA, Bacterial (chemistry, drug effects, genetics, physiology)
  • RNA, Ribosomal, 16S (chemistry, drug effects, genetics, physiology)
  • RNA, Transfer (metabolism)
  • Ribosomes (drug effects, ultrastructure)
  • Tetracycline Resistance (genetics)
  • Tetracyclines (metabolism, pharmacology)
  • Tigecycline

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