PMID:9415442
| Citation |
Mansilla, F, Knudsen, CR, Laurberg, M and Clark, BF (1997) Mutational analysis of Escherichia coli elongation factor Tu in search of a role for the N-terminal region. Protein Eng. 10:927-34 |
|---|---|
| Abstract |
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. |
| Links | |
| Keywords |
DNA Mutational Analysis; Escherichia coli/chemistry; GTP Phosphohydrolase-Linked Elongation Factors/metabolism; Guanosine Diphosphate/metabolism; Guanosine Triphosphate/metabolism; Kinetics; Models, Molecular; Mutagenesis; Nucleic Acid Conformation; Peptide Elongation Factor Tu/chemistry; Peptide Elongation Factor Tu/genetics; Peptide Elongation Factor Tu/metabolism; Protein Conformation; Pyridones/pharmacology; RNA, Transfer, Phe/metabolism |
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