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

Abstract

In our previous work (Mansilla et al. (1997) Protein Eng. 10, 927^934) we showed that Arg U of Escherichia coli elongation factor Tu (EF1A) plays an essential role in aminoacyl-tRNA (aa-tRNA) binding. Substitution of Arg U by Ala or Glu lost this activity. We proposed that Arg U forms a salt bridge with the charged conserved amino acid Glu PUP (Asp PVR 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 Glu PUP to arginine, either alone (Glu PUP Arg), or in combination with one of the above mentioned mutations (Arg U Glu/Glu PUP Arg) in order to test this hypothesis. Our results show that, in confirmation of our thesis based on structural knowledge, the substitution of Glu PUP (Asp PVR ) decreases the ability of EF1A:GTP to bind aa-tRNA.z 1998 Federation of European Biochemical Societies.