In vivo interaction between mutated tryptophan repressors of Escherichia coli.

Abstract:

: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.

journal_name

J Mol Biol

authors

Storbakk N,Fenton C,Riise HM,Nilsen IW,El-Gewely MR

doi

10.1006/jmbi.1996.0135

subject

Has Abstract

pub_date

1996-03-15 00:00:00

pages

889-96

issue

5

eissn

0022-2836

issn

1089-8638

pii

S0022283696901357

journal_volume

256

pub_type

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