biostudies-literaturePolikanov YSHoward Hughes Medical InstituteNational Center of Neurology and PsychiatryMinistry of Education, Culture, Sports, Science and TechnologyNational Institutes of HealthNIGMS NIH HHS541-50https://www.ebi.ac.uk/biostudies/studies/S-EPMC4334386biostudies-literatureUnknown56(4)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 activitMolecular cellNegamycin interferes with decoding and translocation by simultaneous interaction with rRNA and tRNA.PMC433438625650106P01 GM022778GM022778R01 GM106386GM10638623-5Szal TShiozuka MJiang FVazquez-Laslop NMatsuda RSteitz TAMankin ASPolikanov YSGupta PfalseNegamycin interferes with decoding and translocation by simultaneous interaction with rRNA and tRNA.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 activit2014-01-01T00:00:00Z2014 Nov2025-06-28T03:05:18.036Z2025-06-28T03:05:18.036ZS-EPMC43343862530692210.1016/j.molcel.2014.09.021