Mutational analysis of Escherichia coli elongation factor Tu in search of a role for the N-terminal region

作者：Mansilla F; Knudsen CR; Laurberg M; Clark BFC

来源：Protein Engineering, 1997, 10(8): 927-934.

DOI：10.1093/protein/10.8.927

• aminoacyl-tRNA binding
• basic residues
• elongation factor Tu
• mutational analysis
• structural rearrangement

We have mutated lysine 2 and arginine 7 in elongation factor Tu from Escherichia coli separately either to alanine or glutamic acid, The aim of the work was to reveal the possible interactions between the conserved N-terminal part of the molecule, which is rich in basic residues and aminoacyl-tRNA, The enzymatic characterization, comprising GDP and GTP temperature stability assays and measurement of nucleotide dissociation and association rate constants, GTPase activity and aminoacyl-tRNA binding, shows that position 2 is not involved in aminoacyl-tRNA binding, while position 7 is necessary to accomplish this activity. Furthermore, arginine 7 seems to play a role in regulating the binding of GTP. The three-dimensional structure of the ternary complex, EF-Tu:GTP:Phe-tRNA(Phe), involving Thermus aquaticus EF-Tu and yeast Phe-tRNA(Phe), shows that Arg7 is in a position which permits salt bridge formation with Asp284, thus binding the N-terminus tightly to domain 2, We propose that this interaction is needed for aminoacyl-tRNA binding, and also for completing the structural rearrangement, which takes place when the factor switches from its GDP to its GTP form.

• 出版日期1997-8