TY - JOUR
T1 - Negamycin Interferes with Decoding and Translocation by Simultaneous Interaction with rRNA and tRNA
AU - Polikanov, Yury S.
AU - Szal, Teresa
AU - Jiang, Fuyan
AU - Gupta, Pulkit
AU - Matsuda, Ryoichi
AU - Shiozuka, Masataka
AU - Steitz, Thomas A.
AU - Vázquez-Laslop, Nora
AU - Mankin, Alexander S.
N1 - Funding Information:
We thank L. Smith for bringing the NEG project to our attention and for editing the manuscript, P. Moore for valuable discussions and critical reading of the manuscript, D. Wilson and A. Starosta for providing the TetM-encoding plasmid, K. Fredrick for providing plasmid pKF291, R.L. Grodzicki for preparation of the unmodified tRNAs and critical reading of the manuscript, and members of the A.S.M. and T.A.S. laboratories for discussions. We thank the staff at the Advanced Photon Source (beamline 24ID) and at the National Synchrotron Light Source (beamline X25) for help during data collection and the staff at the Richards Center at Yale University for computational support. This work was supported by NIH grants GM106386 (A.S.M. and N.V.-L.) and GM022778 (T.A.S.); an Intramural Research Grant (23-5) for Neurological and Psychiatric Disorder from the National Center of Neurology and Psychiatry; and a Grant-in-Aid #25650106 from Ministry of Education, Culture, Sports, Science and Technology, Japan (R.M.). The SPARK program of the Stevenson High School supported summer internship of F.J.
Publisher Copyright:
© 2014 Elsevier Inc.
PY - 2014/11/20
Y1 - 2014/11/20
N2 - 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 theprimary 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.
AB - 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 theprimary 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.
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U2 - 10.1016/j.molcel.2014.09.021
DO - 10.1016/j.molcel.2014.09.021
M3 - Article
C2 - 25306922
AN - SCOPUS:84912565442
VL - 56
SP - 541
EP - 550
JO - Molecular Cell
JF - Molecular Cell
SN - 1097-2765
IS - 4
ER -