Conformationally Restricted Elongation Factor G Retains GTPase Activity but Is Inactive in Translocation on the Ribosome

• Published in: Molecular cell 2000-08, Vol.6 (2), p.501-505
• Main Authors: Peske, Frank, Matassova, Natalia B., Savelsbergh, Andreas, Rodnina, Marina V., Wintermeyer, Wolfgang
• Format: Article
• Language: English
• Subjects: Amino Acid Substitution; Cross-Linking Reagents; Cysteine; Escherichia coli; Escherichia coli - metabolism; GTP; GTP Phosphohydrolases - metabolism; GTPase; Guanosine Diphosphate - metabolism; Kinetics; Models, Molecular; Mutagenesis, Site-Directed; Peptide Elongation Factor G - chemistry; Peptide Elongation Factor G - metabolism; Protein Conformation; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; Ribosomes - metabolism; RNA, Transfer - metabolism; Thermus thermophilus - metabolism; tRNA
• ISSN: 1097-2765; 1097-4164
• DOI: 10.1016/S1097-2765(00)00049-6

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.