Mutational analysis of Escherichia coli elongation factor Tu in search of a role for the N-terminal region.

Francisco Mansilla, Charlotte Rohde Knudsen, M Laurberg, Brian F. C. Clark

    Research output: Contribution to journal/Conference contribution in journal/Contribution to newspaperJournal articleResearchpeer-review


    We have mutated lysine 2 and arginine 7 in elongation factor Tu from Escherichia coli separately either to alanine or glutamic acid. The aim of the work was to reveal the possible interactions between the conserved N-terminal part of the molecule, which is rich in basic residues and aminoacyl-tRNA. The enzymatic characterization, comprising GDP and GTP temperature stability assays and measurement of nucleotide dissociation and association rate constants, GTPase activity and aminoacyl-tRNA binding, shows that position 2 is not involved in aminoacyl-tRNA binding, while position 7 is necessary to accomplish this activity. Furthermore, arginine 7 seems to play a role in regulating the binding of GTP. The three-dimensional structure of the ternary complex, EF-Tu:GTP:Phe-tRNAPhe, involving Thermus aquaticus EF-Tu and yeast Phe-tRNA(Phe), shows that Arg7 is in a position which permits salt bridge formation with Asp284, thus binding the N-terminus tightly to domain 2. We propose that this interaction is needed for aminoacyl-tRNA binding, and also for completing the structural rearrangement, which takes place when the factor switches from its GDP to its GTP form.
    Udgivelsesdato: 1997-Aug
    Original languageEnglish
    JournalProtein Engineering Design and Selection (Print)
    Pages (from-to)927-34
    Number of pages7
    Publication statusPublished - 1998


    • DNA Mutational Analysis
    • Escherichia coli
    • GTP Phosphohydrolase-Linked Elongation Factors
    • Guanosine Diphosphate
    • Guanosine Triphosphate
    • Kinetics
    • Models, Molecular
    • Mutagenesis
    • Nucleic Acid Conformation
    • Peptide Elongation Factor Tu
    • Protein Conformation
    • Pyridones
    • RNA, Transfer, Phe

    Cite this