Mutational analysis of Glu272in elongation factor 1A of E. coli

In our previous work (Mansilla et al. (1997) Protein Eng. 10, 927–934) we showed that Arg7of Escherichia colielongation factor Tu (EF1A) plays an essential role in aminoacyl‐tRNA (aa‐tRNA) binding. Substitution of Arg7by Ala or Glu lost this activity. We proposed that Arg7forms a salt bridge with the charged conserved amino acid Glu272(Asp284in 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 Glu272to 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.