Functional effects of deleting the coiled-coil motif in Escherichia coli elongation factor Ts.
Elongation factor Ts (EF-Ts) is the guanine nucleotide-exchange factor for elongation factor Tu (EF-Tu) that is responsible for promoting the binding of aminoacyl-tRNA to the mRNA-programmed ribosome. The structure of the Escherichia coli EF-Tu-EF-Ts complex reveals a protruding antiparallel coiled-coil motif in EF-Ts, which is responsible for the dimerization of EF-Ts in the crystal. In this study, the sequence encoding the coiled-coil motif in EF-Ts was deleted from the genome in Escherichia coli by gene replacement. The growth rate of the resulting mutant strain was 70-95% of that of the wild-type strain, depending on the growth conditions used. The mutant strain sensed amino acid starvation and synthesized the nucleotides guanosine 5'-diphosphate 3'-diphosphate and guanosine 5'-triphosphate 3'-diphosphate at a lower cell density than the wild-type strain. Deletion of the coiled-coil motif only partially reduced the ability of EF-Ts to stimulate the guanine nucleotide exchange in EF-Tu. However, the concentration of guanine nucleotides (GDP and GTP) required to dissociate the mutant EF-Tu-EF-Ts complex was at least two orders of magnitude lower than that for the wild-type complex. The results show that the coiled-coil motif plays a significant role in the ability of EF-Ts to compete with guanine nucleotides for the binding to EF-Tu. The present results also indicate that the deletion alters the competition between EF-Ts and kirromycin for the binding to EF-Tu.
Karring
Henrik
H
Department of Molecular Biology, Aarhus University, Denmark.
Björnsson
Asgeir
A
Thirup
Søren
S
Clark
Brian F C
BF
Knudsen
Charlotte R
CR
D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D004926:Escherichia coli / Q000737:chemistry / Q000378:metabolism*; D006150:Guanine Nucleotides / Q000378:metabolism; D008958:Models, Molecular; D010445:Peptide Elongation Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation
D017931:DNA Primers; D006150:Guanine Nucleotides; D010445:Peptide Elongation Factors; C023512:elongation factor Ts