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 promotor/o perator, thus putting the beta-galactosidase activity under the contro l of the Trp repressor, we can determine quantitatively the relative r epression activity of such mutant(s). We used this technique to analys e the biological consequences of substituting certain amino acid resid ues in only one of the two corepressor binding pockets. By combining t wo compatible plasmids in this strain, one expressing the mutant T44M and the other expressing only one substitution at a time at position 8 5, we analysed the repression activity of the resulting interactions i n vivo. This approach allowed us to engineer active dimer repressors m ade of two inactive or partially active monomers. Amino acid substitut ions at position 85 with a positive or with an indole ring (W) appeare d to complement T44M, while amino acids with a negative charge did not . Only L substitution at position 85 appeared to restore activity amon g the hydrophobic amino acids tested. Similar to the wild-type repress or activity, the successful mutant-mutant interactions were L-tryptoph an dependent. In vivo regulation by three known L-tryptophan analogues demonstrated the same trend of regulation among the wild-type repress ors and the active mutant-mutant combinations. (C) 1996 Academic Press Limited