title: Minor groove recognition is important for the transcription factor PhoB: a surface plasmon resonance study.
abstract: The two-component regulatory system PhoR/PhoB induces the expression of several genes in response to phosphate starvation in Escherichia coli. In order to quantify these protein-DNA interactions and to study the time-resolved dynamics of the binding mechanism, the specific recognition of different oligonucleotide duplexes by the DNA-binding domain of PhoB (PhoB(DBD)) was analyzed using surface plasmon resonance. In addition the two point mutants PhoB(DBD)D196A and PhoB(DBD)R219A were obtained and the DNA recognition in comparison to the wildtype PhoB(DBD) was investigated. Aspartic acid 196 and arginine 219 mediate specific minor groove interactions. All results reveal that at high PhoB(DBD)-concentrations all recognition sequences of the pho box are occupied. Decreasing the protein amount results in a mixture of free oligonucleotides and DNA molecules occupied by two WT-PhoB(DBD). Moreover, the SPR results indicate that both binding site segments, the TGTCA-motif and the A/T-rich minor groove, are essential for the binding process. A comparison of different regulons additionally proved the dependency of the recognition process on the base composition of the minor groove.
authors.affiliation: Organic and Bioorganic Chemistry, Bielefeld University, PO Box 100131, 33501 Bielefeld, Germany.
authors.forename: M
authors.initials: M
authors.lastname: Ritzefeld
authors.forename: K
authors.initials: K
authors.lastname: Wollschläger
authors.forename: G
authors.initials: G
authors.lastname: Niemann
authors.forename: D
authors.initials: D
authors.lastname: Anselmetti
authors.forename: N
authors.initials: N
authors.lastname: Sewald
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D001665:Binding Sites; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D010710:Phosphates / Q000378:metabolism; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics; D018085:Regulon; D020349:Surface Plasmon Resonance; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D010710:Phosphates; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; C061278:PhoB protein, Bacteria; C061279:PhoR protein, Bacteria