NUTATIONAL ANALYSIS OF GLU(272) IN ELONGATION-FACTOR 1A OF ESCHERICHIA-COLI

In our previous work (Mansilla et al, (1997) Protein Eng, 10, 927-934) we showed that Arg(7) of Escherichia coli elongation factor Tu (EF1A) plays an essential role in aminoacyl-tRNA (aa-tRNA) binding. Substitu tion of Arg(7) by Ala or Glu lost this activity. We proposed that Arg7 forms a salt bridge with the charged conserved amino acid Glu(272) (A sp(284) in Thermus aquaticus) thereby binding the N-terminal region of the protein to domain 2 and thus completing the conformational rearra ngement needed for binding aa-tRNA. In this work we have mutated Glu(2 72) to arginine, either alone (Glu(272)Arg), Or in combination with on e of the above mentioned mutations (Arg(7)Glu/Glu(272)Arg) in order to test this hypothesis. Our results show that, in confirmation of our t hesis based on structural knowledge, the substitution of Glu(272) (Asp (284)) decreases the ability of EF1A:GTP to bind aa-tRNA. (C) 1998 Fed eration of European Biochemical Societies.