title: Trp repressor-operator binding: NMR and electrophoretic mobility shift studies of the effect of DNA sequence and corepressor binding on two Trp repressor-operator complexes.
abstract: In Trp repressor-DNA complexes, most interactions either occur with phosphate groups or are water-mediated hydrogen bonds to bases. To examine the factors involved in DNA selectivity, we have studied Trp repressor binding to two operator sequences, trpR(S)() and trpO(M)(), with L-tryptophan or 5-methyltryptophan as corepressor. These operators contain all the consensus bases but differ at base pairs contacted by their phosphate groups. In electrophoretic mobility shift assays (EMSAs) the trpR(S)() sequence gives solely 1:1 protein-DNA complexes with either corepressor. The trpO(M )()sequence binds more weakly than trpR(S)(). It gives dissociating 2:1 complexes in EMSAs with L-tryptophan, but both 1:1 and 2:1 complexes are observed with 5-methyltryptophan or if glycerol is present in the gel. The backbone resonances of the TrpR-L-tryptophan-DNA complexes were assigned using triple-resonance experiments and selectively (15)N labeled protein. On changing the DNA sequence, the largest differences in the NMR spectra are at residues 78-81, at the turn of the helix-turn-helix motif and the tip of the recognition helix. I79 and A80 interact with the conserved bases of the operators, while G78 and T81 interact with phosphate groups at bases that differ between the two sequences. Changing the corepressor from L-tryptophan to 5-methyltryptophan causes effects at residues 52, 60, 61, and 85, which do not interact with the DNA. The spectra suggest that there is mutual induced fit between protein and DNA so that sequence changes at bases contacted only by the phosphate groups affect the environment of the protein at residues that bind to conserved bases elsewhere in the DNA.
authors.affiliation: School of Biosciences, University of Birmingham, Edgbaston, UK.
authors.forename: Mahesh
authors.initials: M
authors.lastname: Jaseja
authors.forename: Mark
authors.initials: M
authors.lastname: Jeeves
authors.forename: Eva I
authors.initials: EI
authors.lastname: Hyde
mesh_terms: D001426:Bacterial Proteins; D001665:Binding Sites; D004269:DNA, Bacterial / Q000737:chemistry* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay / Q000379:methods; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D046911:Macromolecular Substances; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D009875:Operator Regions, Genetic; D012097:Repressor Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D014364:Tryptophan / Q000737:chemistry* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D046911:Macromolecular Substances; D012097:Repressor Proteins; C027190:TRPR protein, E coli; D014364:Tryptophan