Modulation of the Escherichia coli tryptophan repressor protein by engineered peptides.
We have used the E. coli tryptophan repressor (TrpR) as a model protein for modulation by engineered peptides both in vivo and in vitro. The tryptophan operator promoter-lacZ reporter system was used to investigate the in vivo ability of several synthetic peptides to modulate TrpR function. GMSA (gel mobility shift analysis) was used to study the in vitro ability of the peptides to modulate binding of the TrpR protein to the operator DNA. Peptides WRW, DRW, DW, RW enhanced TrpR binding to the operator in vivo at 100 microM 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 than corepressor. The peptide WR could neither enhance nor impede binding between TrpR and the operator in vivo or in vitro, suggesting that the presence of a carboxyl tryptophan residue may be necessary for binding to the TrpR protein. Thin layer chromatography was used to ensure that the peptides had not been subject to proteolysis during the in vitro gel mobility shift assays.
Fenton
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Department of Biotechnology, University of Tromsø, Norway.
Hansen
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A
el-Gewely
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D001426:Bacterial Proteins; D004269:DNA, Bacterial / Q000378:metabolism; D004151:Dipeptides / Q000138:chemical synthesis / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects*; D017930:Genes, Reporter; D009842:Oligopeptides / Q000138:chemical synthesis / Q000494:pharmacology*; D009875:Operator Regions, Genetic; D011485:Protein Binding / Q000187:drug effects; D012097:Repressor Proteins / Q000378:metabolism*; D014364:Tryptophan / Q000494:pharmacology*; D001616:beta-Galactosidase / Q000096:biosynthesis; D029968:Escherichia coli Proteins
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004151:Dipeptides; D009842:Oligopeptides; D012097:Repressor Proteins; D014364:Tryptophan; D001616:beta-Galactosidase; C027190:TRPR protein, E coli; D029968:Escherichia coli Proteins