Properties of a Genetically Engineered G Domain of Elongation Factor Tu

• Published in: Proceedings of the National Academy of Sciences - PNAS 1987-05, Vol.84 (10), p.3141-3145
• Main Authors: Parmeggiani, Andrea, Guido W. M. Swart, Mortensen, Kim K., Jensen, Michael, Brian F. C. Clark, Dente, Luciana, Cortese, Riccardo
• Format: Article
• Language: English
• Subjects: Binding Sites; Biochemistry; Biological and medical sciences; DNA; Escherichia coli; Escherichia coli - genetics; Escherichia coli - metabolism; Fundamental and applied biological sciences. Psychology; Genetic Engineering; Gross domestic product; GTP Phosphohydrolase-Linked Elongation Factors - metabolism; Guanine nucleotides; Guanosine Diphosphate - metabolism; Hydrolysis; Ligands; Molecular and cellular biology; Molecular genetics; Molecules; Peptide Elongation Factor Tu - genetics; Peptide Elongation Factor Tu - metabolism; Peptide elongation factors; Protein Binding; Protein Conformation; Proteins; Ribosomes; Translation. Translation factors. Protein processing
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.84.10.3141

The G domain of elongation factor Tu (EF-Tu), representing the N-terminal half of the factor according to its three-dimensional model traced at high resolution, has been isolated by genetic manipulation of tufA and purified to homogeneity. The G domain, whose primary structure shares homology with the eukaryotic protein p21, is capable of supporting the basic activities of the intact molecule (guanine nucleotide binding in 1:1 molar ratio and GTPase activity). However, it is no longer exposed to the allosteric mechanisms regulating EF-Tu. The G-domain complexes with GTP and GDP display similar Kd ′values in the μ M range, in contrast to EF-Tu that binds GDP much more tightly than GTP. Its GTPase shows the characteristics of a slow turnover reaction (0.1 mmol· sec-1· mol-1of G domain), whose rate closely corresponds to the initial hydrolysis rate of EF-Tu· GTP in the absence of effectors and lies in the typical range of GTPase of the p21 protein. Of the EF-Tu ligands only the ribosome displays a clear effect enhancing the G-domain GTPase. Our results suggest that the middle and C-terminal domain play an essential role in regulating the activity of the N-terminal domain of the intact molecule as well as in the interactions of EF-Tu with aminoacylated tRNA, elongation factor Ts, and kirromycin. With the isolation of the G domain of EF-Tu, a model protein has been constructed for studying and comparing common characteristics of the guanine nucleotide-binding proteins.