MODULATION OF THE ESCHERICHIA-COLI TRYPTOPHAN REPRESSOR PROTEIN BY ENGINEERED PEPTIDES

We have used the E. coil tryptophan repressor (TrpR) as a model protei n for modulation by engineered peptides both in vivo and in vitro. The tryptophan operator promoter-lacZ reporter system was used to investi gate the in vivo ability of several synthetic peptides to modulate Trp R function. GMSA (gel mobility shift analysis) was used to study the i n vitro ability of the peptides to modulate binding of the TrpR protei n to the operator DNA. Peptides WRW, DRW, DW, RW enhanced TrpR binding to the operator in vivo at 100 mu M concentrations. The same peptides enhanced TrpR binding to the operator in vitro at 1 mM concentrations . The peptide RRW reduced TrpR binding to the tryptophan operator both in vivo and in vitro. Thus the peptide RRW acted more as an inducer t han corepressor. The peptide WR could neither enhance nor impede bindi ng between TrpR and the operator in vivo or in vitro, suggesting that the presence of a carboxyl tryptophan residue may be necessary for bin ding to the TrpR protein. Thin layer chromatography was used to ensure that the peptides had not been subject to proteolysis during the in v itro gel mobility shift assays. (C) 1998 Academic Press.