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.
Peske, F., Matassova, N. B., Savelsbergh, A., Rodnina, M. V., & Wintermeyer, W. (2000). Conformationally restricted elongation factor G retains GTPase activity but is inactive in translocation on the ribosome. Molecular Cell, 6(2), 501–505. https://doi.org/10.1016/S1097-2765(00)00049-6