In vivo interaction between mutated tryptophan repressors of Escherichia coli.
By expressing a mutant trpR gene in an Escherichia coli strain that is trpR and has beta-galactosidase activity fused to the trp promoter/operator, thus putting the beta-galactosidase activity under the control of the Trp repressor, we can determine quantitatively the relative repression activity of such mutant(s). We used this technique to analyse the biological consequences of substituting certain amino acid residues in only one of the two corepressor binding pockets. By combining two compatible plasmids in this strain, one expressing the mutant T44M and the other expressing only one substitution at a time at position 85, we analysed the repression activity of the resulting interactions in vivo. This approach allowed us to engineer active dimer repressors made of two inactive or partially active monomers. Amino acid substitutions at position 85 with a positive or with an indole ring (W) appeared to complement T44M, which amino acids with a negative charge did not. Only L substitution at position 85 appeared to restore activity among the hydrophobic amino acids tested. Similar to the wild-type repressor activity, the successful mutant-mutant interactions were L-tryptophan dependent. In vivo regulation by three known L-tryptophan analogues demonstrated the same trend of regulation among the wild-type repressors and the active mutant-mutant combinations.
Storbakk
N
N
Department of Biotechnology, Institute of Medical Biology, University of Tromsø, Norway.
Fenton
C
C
Riise
H M
HM
Nilsen
I W
IW
El-Gewely
M R
MR
D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D012097:Repressor Proteins / Q000235:genetics* / Q000378:metabolism*; D014364:Tryptophan / Q000031:analogs & derivatives / Q000378:metabolism*; D001616:beta-Galactosidase / Q000235:genetics; D029968:Escherichia coli Proteins
D001426:Bacterial Proteins; D017931:DNA Primers; D004269:DNA, Bacterial; D012097:Repressor Proteins; D014364:Tryptophan; D001616:beta-Galactosidase; C027190:TRPR protein, E coli; D029968:Escherichia coli Proteins