title: Recognition of binding sites I and II by the TrpI activator protein of pseudomonas aeruginosa: efficient binding to both sites requires InGP even when site II is replaced by site I.
abstract: TrpI protein, the activator of transcription of the trpBA operon of three species of fluorescent Pseudomonads, bends the DNA when it forms either of two well-characterized complexes with the trpBA regulatory region. In complex 1, TrpI is bound only to its strong binding site (site I), whereas in complex 2, which is required for activation of the trpBA promoter, TrpI is bound both to site I and to the weaker site II. Indoleglycerol phosphate (InGP) strongly stimulates formation of complex 2 and is required for activation. The present study focuses on the binding of TrpI to DNA containing a duplication of site I and the effect of the duplication on TrpI-induced DNA bending. We find that even on DNA containing a tandem (direct or inverted) duplication of site I, the formation of DNA-TrpI complexes with both sites occupied is strongly stimulated by InGP. Thus, even when TrpI binding to two adjacent sites needs not be cooperative, InGP significantly promotes the formation of complex 2. Gel binding data indicate that InGP can have several effects: (1) TrpI molecules bound to either of two adjacent strong binding sites appear to interfere with binding to the other site; InGP relieves this apparent interference. (2) InGP increases the intrinsic affinity of TrpI for sites I and II and/or enhances cooperative TrpI binding to adjacent DNA sites. Furthermore, a third molecule of TrpI can form a footprint adjacent to the duplication on DNA containing a direct (but not inverted) repeat of site I, indicating that TrpI bound to site I is oriented asymmetrically in spite of the quasi-symmetry of the binding site. The calculated bending angle for DNA in complex 2 is increased by approximately 20 degrees when site I is substituted in either orientation for site II; thus, on DNA containing a site I duplication, the bending angle of complex 2 is nearly twice that of complex 1.
authors.affiliation: Department of Biological Sciences, University of Iowa, Iowa City, IA 52242, USA.
authors.forename: I
authors.initials: I
authors.lastname: Olekhnovich
authors.forename: G N
authors.initials: GN
authors.lastname: Gussin
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D018983:DNA Footprinting; D004269:DNA, Bacterial / Q000378:metabolism; D004268:DNA-Binding Proteins; D003850:Deoxyribonuclease I; D015964:Gene Expression Regulation, Bacterial; D005994:Glycerophosphates / Q000378:metabolism*; D008969:Molecular Sequence Data; D011550:Pseudomonas aeruginosa / Q000235:genetics* / Q000378:metabolism; D013698:Templates, Genetic; D015534:Trans-Activators
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D005994:Glycerophosphates; D015534:Trans-Activators; C008117:indoleglycerol phosphate; D003850:Deoxyribonuclease I