MUTATIONAL ANALYSIS OF ESCHERICHIA-COLI ELONGATION-FACTOR TU IN SEARCH OF A ROLE FOR THE N-TERMINAL REGION

We have mutated lysine 2 and arginine 7 in elongation factor Tu from E scherichia coli separately either to alanine or glutamic acid, The aim of the work was to reveal the possible interactions between the conse rved 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 dissoc iation and association rate constants, GTPase activity and aminoacyl-t RNA binding, shows that position 2 is not involved in aminoacyl-tRNA b inding, while position 7 is necessary to accomplish this activity. Fur thermore, arginine 7 seems to play a role in regulating the binding of GTP. The three-dimensional structure of the ternary complex, EF-Tu:GT P:Phe-tRNA(Phe), involving Thermus aquaticus EF-Tu and yeast Phe-tRNA( Phe), shows that Arg7 is in a position which permits salt bridge forma tion with Asp284, thus binding the N-terminus tightly to domain 2, We propose that this interaction is needed for aminoacyl-tRNA binding, an d also for completing the structural rearrangement, which takes place when the factor switches from its GDP to its GTP form.