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Comparing the Affinity of GTPase-binding Proteins using Competition Assays
Published on: October 8, 2015
Conformationally restricted elongation factor G retains GTPase activity but is inactive in translocation on the
F Peske1, N B Matassova, A Savelsbergh
1Institute of Molecular Biology, University of Witten/Herdecke, Witten, Germany.
Abstract:
Elongation factor G (EF-G) from Escherichia coli is a large, five-domain GTPase that promotes tRNA translocation on the ribosome. Full activity requires GTP hydrolysis, suggesting that a conformational change of the factor is important for function. To restrict the intramolecular mobility, two cysteine residues were engineered into domains 1 and 5 of EF-G that spontaneously formed a disulfide cross-link. Cross-linked EF-G retained GTPase activity on the ribosome, whereas it was inactive in translocation as well as in turnover. Both activities were restored when the cross-link was reversed by reduction. These results strongly argue against a GTPase switch-type model of EF-G function and demonstrate that conformational mobility is an absolute requirement for EF-G function on the ribosome.
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