title: The basis for the super-repressor phenotypes of the AV77 and EK18 mutants of trp repressor.
abstract: The DNA-binding properties of two super-repressor mutants of the Escherichia coli trp repressor, EK18 and AV77, have been investigated using steady-state fluorescence anisotropy measurements, in order to further elucidate the basis for their super-repressor phenotypes. Several suggestions have been previously proposed as the basis for the super-repressor phenotype of EK18 and AV77. For the negative to positive charge change EK18 mutant, increased electrostatic interactions between the EK18 mutant and the operator and increased protein-protein interactions between EK18 dimers have been suggested as contributing to the super-repressor phenotype of this mutant. We show that EK18 dimers actually bind to wild-type and variant operator sequences with a decrease in apparent cooperativity and an increase in affinity, compared to WTTR dimers. Thus, the EK18 super-repressor phenotype is not due to increased cooperative binding between EK18 dimers. These results support the hypothesis that the super-repressor phenotype of EK18 arises from increased electrostatic interactions between the mutant and DNA. In the case of the AV77 mutant, weaker binding affinity of apo-AV77 to non-specific DNA, increased selectivity of binding of AV77 for the operator, and a higher population of folded functional AV77 dimers available to bind the operator under limiting L-Trp conditions in vivo, have been proposed for the super-repressor phenotype of this mutant. We show that like the EK18 mutant, apoAV77 binds with higher affinity to non-specific DNA compared to apo-WTTR and that the holo-AV77 mutant does not bind with higher selectivity to the operator, has had been previously proposed. We therefore conclude that the super-repressor phenotype of the AV77 mutant is due to an increase in the population of folded, functional AV77 dimers, under limiting L-Trp conditions in vivo.
authors.affiliation: School of Pharmacy, University of Wisconsin-Madison, 425 N. Charter St, Madison, WI 53706, USA.
authors.forename: A O
authors.initials: AO
authors.lastname: Grillo
authors.forename: C A
authors.initials: CA
authors.lastname: Royer
mesh_terms: D001059:Apoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019281:Dimerization; D004926:Escherichia coli; D019793:Fluorescein / Q000378:metabolism; D005454:Fluorescence Polarization; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics*; D009838:Oligodeoxyribonucleotides / Q000235:genetics / Q000378:metabolism; D009875:Operator Regions, Genetic / Q000235:genetics; D009876:Operon / Q000235:genetics; D010641:Phenotype; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D055672:Static Electricity; D013816:Thermodynamics; D014364:Tryptophan / Q000378:metabolism; D029968:Escherichia coli Proteins
chemical_list: D001059:Apoproteins; D001426:Bacterial Proteins; D004247:DNA; D004268:DNA-Binding Proteins; D019793:Fluorescein; D009838:Oligodeoxyribonucleotides; D012097:Repressor Proteins; D014364:Tryptophan; C027190:TRPR protein, E coli; D029968:Escherichia coli Proteins