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EF-G-dependent GTPase on the ribosome. conformational change and fusidic acid inhibition. Biochemistry 2006 Feb 28;45(8):2504-14

Date

02/24/2006

Pubmed ID

16489743

DOI

10.1021/bi0516677

Scopus ID

2-s2.0-33644542727   67 Citations

Abstract

Protein synthesis studies increasingly focus on delineating the nature of conformational changes occurring as the ribosome exerts its catalytic functions. Here, we use FRET to examine such changes during single-turnover EF-G-dependent GTPase on vacant ribosomes and to elucidate the mechanism by which fusidic acid (FA) inhibits multiple-turnover EF-G.GTPase. Our measurements focus on the distance between the G' region of EF-G and the N-terminal region of L11 (L11-NTD), located within the GTPase activation center of the ribosome. We demonstrate that single-turnover ribosome-dependent EF-G GTPase proceeds according to a kinetic scheme in which rapid G' to L11-NTD movement requires prior GTP hydrolysis and, via branching pathways, either precedes P(i) release (major pathway) or occurs simultaneously with it (minor pathway). Such movement retards P(i) release, with the result that P(i) release is essentially rate-determining in single-turnover GTPase. This is the most significant difference between the EF-G.GTPase activities of vacant and translocating ribosomes [Savelsbergh, A., Katunin, V. I., Mohr, D., Peske, F., Rodnina, M. V., and Wintermeyer, W. (2003) Mol. Cell 11, 1517-1523], which are otherwise quite similar. Both the G' to L11-NTD movement and P(i) release are strongly inhibited by thiostrepton but not by FA. Contrary to the standard view that FA permits only a single round of GTP hydrolysis [Bodley, J. W., Zieve, F. J., and Lin, L. (1970) J. Biol. Chem. 245, 5662-5667], we find that FA functions rather as a slow inhibitor of EF-G.GTPase, permitting a number of GTPase turnovers prior to complete inhibition while inducing a closer approach of EF-G to the GAC than is seen during normal turnover.

Author List

Seo HS, Abedin S, Kamp D, Wilson DN, Nierhaus KH, Cooperman BS

Author

Sameem Abedin MD Assistant Professor in the Medicine department at Medical College of Wisconsin




MESH terms used to index this publication - Major topics in bold

Cryoelectron Microscopy
Dose-Response Relationship, Drug
Escherichia coli
Fluorescence Resonance Energy Transfer
Fusidic Acid
GTP Phosphohydrolases
Guanosine Triphosphate
Hydrolysis
Kinetics
Models, Molecular
Peptide Elongation Factor G
Phosphates
Protein Binding
Protein Conformation
Ribosomes
Thiostrepton
Time Factors
Translocation, Genetic