Mutational analysis of Glu272 in elongation factor 1A of E. coli.
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.
Mansilla
F
F
Institute of Molecular and Structural Biology, Aarhus University, Aarhus C, Denmark.
Knudsen
C R
CR
Clark
B F
BF
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D004252:DNA Mutational Analysis; D004926:Escherichia coli; D018698:Glutamic Acid; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D010441:Peptide Chain Elongation, Translational; D010444:Peptide Elongation Factor Tu / Q000235:genetics / Q000378:metabolism*; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism*
D001426:Bacterial Proteins; D012346:RNA, Transfer, Amino Acyl; D006153:Guanosine Diphosphate; D018698:Glutamic Acid; D006160:Guanosine Triphosphate; D010444:Peptide Elongation Factor Tu