Mutational analysis of Glu272 in elongation factor 1A of E. coli.

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

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    Abstract

    In our previous work (Mansilla et al. (1997) Protein Eng. 10, 927-934) we showed that Arg7 of Escherichia coli elongation factor Tu (EF1A) plays an essential role in aminoacyl-tRNA (aa-tRNA) binding. Substitution of Arg7 by Ala or Glu lost this activity. We proposed that Arg7 forms a salt bridge with the charged conserved amino acid Glu272 (Asp284 in Thermus aquaticus) thereby binding the N-terminal region of the protein to domain 2 and thus completing the conformational rearrangement needed for binding aa-tRNA. In this work we have mutated Glu272 to arginine, either alone (Glu272Arg), or in combination with one of the above mentioned mutations (Arg7Glu/Glu272Arg) in order to test this hypothesis. Our results show that, in confirmation of our thesis based on structural knowledge, the substitution of Glu272 (Asp284) decreases the ability of EF1A:GTP to bind aa-tRNA.
    Udgivelsesdato: 1998-Jun-16
    Original languageEnglish
    JournalF E B S Letters
    Volume429
    Issue3
    Pages (from-to)417-20
    Number of pages3
    ISSN0014-5793
    Publication statusPublished - 1998

    Keywords

    • Bacterial Proteins
    • DNA Mutational Analysis
    • Escherichia coli
    • Glutamic Acid
    • Guanosine Diphosphate
    • Guanosine Triphosphate
    • Mutagenesis, Site-Directed
    • Peptide Chain Elongation, Translational
    • Peptide Elongation Factor Tu
    • RNA, Transfer, Amino Acyl

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