TY - JOUR
T1 - Mutational analysis of Escherichia coli elongation factor Tu in search of a role for the N-terminal region.
AU - Mansilla, Francisco
AU - Knudsen, Charlotte Rohde
AU - Laurberg, M
AU - Clark, Brian F. C.
PY - 1998
Y1 - 1998
N2 - 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.
Udgivelsesdato: 1997-Aug
AB - 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.
Udgivelsesdato: 1997-Aug
KW - DNA Mutational Analysis
KW - Escherichia coli
KW - GTP Phosphohydrolase-Linked Elongation Factors
KW - Guanosine Diphosphate
KW - Guanosine Triphosphate
KW - Kinetics
KW - Models, Molecular
KW - Mutagenesis
KW - Nucleic Acid Conformation
KW - Peptide Elongation Factor Tu
KW - Protein Conformation
KW - Pyridones
KW - RNA, Transfer, Phe
M3 - Journal article
C2 - 9415442
SN - 1741-0126
VL - 10
SP - 927
EP - 934
JO - Protein Engineering Design and Selection (Print)
JF - Protein Engineering Design and Selection (Print)
IS - 8
ER -