Thiostrepton inhibits the turnover but not the GTPase of elongation factor G on the ribosome.
The region around position 1067 in domain II of 23S rRNA frequently is referred to as the GTPase center of the ribosome. The notion is based on the observation that the binding of the antibiotic thiostrepton to this region inhibited GTP hydrolysis by elongation factor G (EF-G) on the ribosome at the conditions of multiple turnover. In the present work, we have reanalyzed the mechanism of action of thiostrepton. Results obtained by biochemical and fast kinetic techniques show that thiostrepton binding to the ribosome does not interfere with factor binding or with single-round GTP hydrolysis. Rather, the antibiotic inhibits the function of EF-G in subsequent steps, including release of inorganic phosphate from EF-G after GTP hydrolysis, tRNA translocation, and the dissociation of the factor from the ribosome, thereby inhibiting the turnover reaction. Structurally, thiostrepton interferes with EF-G footprints in the alpha-sarcin stem loop (A2660, A2662) located in domain VI of 23S rRNA. The results indicate that thiostrepton inhibits a structural transition of the 1067 region of 23S rRNA that is important for functions of EF-G after GTP hydrolysis.
Rodnina
M V
MV
Institute of Molecular Biology, University of Witten/Herdecke, D-58448 Witten, Germany.
Savelsbergh
A
A
Matassova
N B
NB
Katunin
V I
VI
Semenkov
Y P
YP
Wintermeyer
W
W
D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D020963:GTP Phosphohydrolase-Linked Elongation Factors / Q000378:metabolism*; D006160:Guanosine Triphosphate; D007700:Kinetics; D020653:Peptide Elongation Factor G; D010445:Peptide Elongation Factors / Q000378:metabolism*; D010710:Phosphates / Q000378:metabolism; D012338:RNA, Ribosomal, 23S / Q000378:metabolism; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism*; D013883:Thiostrepton / Q000494:pharmacology*; D014178:Translocation, Genetic
D020653:Peptide Elongation Factor G; D010445:Peptide Elongation Factors; D010710:Phosphates; D012338:RNA, Ribosomal, 23S; D012346:RNA, Transfer, Amino Acyl; C019037:tRNA, peptidyl-; D006160:Guanosine Triphosphate; D020963:GTP Phosphohydrolase-Linked Elongation Factors; D013883:Thiostrepton