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 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-tRNAPhe, 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.
Mansilla
F
F
Institute of Molecular and Structural Biology, Aarhus University, Aarhus C, Denmark.
Knudsen
C R
CR
Laurberg
M
M
Clark
B F
BF
D004252:DNA Mutational Analysis; D004926:Escherichia coli / Q000737:chemistry*; D020963:GTP Phosphohydrolase-Linked Elongation Factors / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D016296:Mutagenesis; D009690:Nucleic Acid Conformation; D010444:Peptide Elongation Factor Tu / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D011487:Protein Conformation; D011728:Pyridones / Q000494:pharmacology; D012360:RNA, Transfer, Phe / Q000378:metabolism
D011728:Pyridones; D012360:RNA, Transfer, Phe; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D020963:GTP Phosphohydrolase-Linked Elongation Factors; D010444:Peptide Elongation Factor Tu; C008536:mocimycin