title: Negamycin interferes with decoding and translocation by simultaneous interaction with rRNA and tRNA.
abstract: Negamycin (NEG) is a ribosome-targeting antibiotic that exhibits clinically promising activity. Its binding site and mode of action have remained unknown. We solved the structure of the Thermus thermophilus ribosome bound to mRNA and three tRNAs, in complex with NEG. The drug binds to both small and large ribosomal subunits at nine independent sites. Resistance mutations in the 16S rRNA unequivocally identified the binding site in the vicinity of the conserved helix 34 (h34) in the small subunit as the primary site of antibiotic action in the bacterial and, possibly, eukaryotic ribosome. At this site, NEG contacts 16S rRNA as well as the anticodon loop of the A-site tRNA. Although the NEG site of action overlaps with that of tetracycline (TET), the two antibiotics exhibit different activities: while TET sterically hinders binding of aminoacyl-tRNA to the ribosome, NEG stabilizes its binding, thereby inhibiting translocation and stimulating miscoding.
authors.affiliation: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520, USA; Howard Hughes Medical Institute, Yale University, New Haven, CT 06520, USA.
authors.forename: Yury S
authors.initials: YS
authors.lastname: Polikanov
authors.affiliation: Center for Pharmaceutical Biotechnology, University of Illinois at Chicago, Chicago, IL 60607, USA.
authors.forename: Teresa
authors.initials: T
authors.lastname: Szal
authors.affiliation: Center for Pharmaceutical Biotechnology, University of Illinois at Chicago, Chicago, IL 60607, USA; Adlai E. Stevenson High School, Lincolnshire, IL 60069, USA.
authors.forename: Fuyan
authors.initials: F
authors.lastname: Jiang
authors.affiliation: Center for Pharmaceutical Biotechnology, University of Illinois at Chicago, Chicago, IL 60607, USA.
authors.forename: Pulkit
authors.initials: P
authors.lastname: Gupta
authors.affiliation: Department of Life Sciences, The University of Tokyo at Komaba, Komaba, 153-8902, Japan.
authors.forename: Ryoichi
authors.initials: R
authors.lastname: Matsuda
authors.affiliation: Department of Life Sciences, The University of Tokyo at Komaba, Komaba, 153-8902, Japan.
authors.forename: Masataka
authors.initials: M
authors.lastname: Shiozuka
authors.affiliation: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520, USA; Howard Hughes Medical Institute, Yale University, New Haven, CT 06520, USA. Electronic address: thomas.steitz@yale.edu.
authors.forename: Thomas A
authors.initials: TA
authors.lastname: Steitz
authors.affiliation: Center for Pharmaceutical Biotechnology, University of Illinois at Chicago, Chicago, IL 60607, USA. Electronic address: nvazquez@uic.edu.
authors.forename: Nora
authors.initials: N
authors.lastname: Vázquez-Laslop
authors.affiliation: Center for Pharmaceutical Biotechnology, University of Illinois at Chicago, Chicago, IL 60607, USA.
authors.forename: Alexander S
authors.initials: AS
authors.lastname: Mankin
mesh_terms: D020816:Amino Acid Motifs; D000599:Amino Acids, Diamino / Q000737:chemistry; D000900:Anti-Bacterial Agents / Q000737:chemistry*; D001483:Base Sequence; D001665:Binding Sites; D018360:Crystallography, X-Ray; D008958:Models, Molecular; D014176:Protein Biosynthesis; D011500:Protein Synthesis Inhibitors / Q000737:chemistry*; D020871:RNA Stability; D012329:RNA, Bacterial / Q000737:chemistry*; D012333:RNA, Messenger / Q000737:chemistry; D012335:RNA, Ribosomal / Q000737:chemistry*; D012343:RNA, Transfer / Q000737:chemistry*; D054681:Ribosome Subunits, Large, Bacterial / Q000737:chemistry; D054680:Ribosome Subunits, Small, Bacterial / Q000737:chemistry; D016963:Thermus thermophilus
chemical_list: D000599:Amino Acids, Diamino; D000900:Anti-Bacterial Agents; D011500:Protein Synthesis Inhibitors; D012329:RNA, Bacterial; D012333:RNA, Messenger; D012335:RNA, Ribosomal; D012343:RNA, Transfer; C100200:negamycin