title: Establishment of lysogeny in bacteriophage 186. DNA binding and transcriptional activation by the CII protein.
abstract: The CII protein of bacteriophage 186 is a transcriptional activator of the helix-turn helix family required for establishment of the lysogenic state. DNA binding by 186 CII is unusual in that the invertedly repeated half sites are separated by 20 base pairs, or two turns of the DNA helix, rather than the one turn usually associated with this class of proteins. Here, we investigate quantitatively the DNA binding properties of CII and its interaction with RNA polymerase at the establishment promoter, p(E). The stoichiometry of CII binding was determined by sedimentation equilibrium experiments using a fluorescein-labeled oligonucleotide and purified CII. These experiments indicate that the CII species bound to DNA is a dimer, with additional weak binding of a tetrameric species at high concentrations. Examination of the thermodynamic linkages between CII self-association and DNA binding shows that CII binds to the DNA as a preformed dimer (binding free energy, 9.9 kcal/mol at 4 degrees C) rather than by association of monomers on the DNA. CII binding induces in the DNA a bend of 41 (+/- 5) degrees. The spacing between the binding half sites was shown to be important for CII binding, insertion or removal of just 1 base pair significantly reducing the affinity for CII. Removal of 5 or 10 base pairs between binding half sites eliminated binding, as did insertion of an additional 10 base pairs. CII binding at p(E) was improved marginally by the presence of RNA polymerase (DeltaDeltaG = -0.5 (+/- 0.3) kcal/mol). In contrast, the binding of RNA polymerase at p(E) was undetectable in the absence of CII but was improved markedly by the presence of CII. Thus, CII appears to recruit RNA polymerase to the promoter. The nature of the base pair changes in mutant phage, selected by their inability to establish lysogeny, are consistent with this mechanism of CII action.
authors.affiliation: Biochemistry Department, University of Adelaide, Adelaide, Australia 5001. keith.shearwin@adelaide.edu.au
authors.forename: K E
authors.initials: KE
authors.lastname: Shearwin
authors.forename: J B
authors.initials: JB
authors.lastname: Egan
mesh_terms: D001483:Base Sequence; D001665:Binding Sites; D003090:Coliphages / Q000235:genetics*; D004247:DNA / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D008242:Lysogeny; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D013816:Thermodynamics; D014157:Transcription Factors / Q000378:metabolism*; D015533:Transcriptional Activation; D014764:Viral Proteins
chemical_list: D014157:Transcription Factors; D014764:Viral Proteins; C039000:cII protein, bacteriophage lambda; D004247:DNA; D012321:DNA-Directed RNA Polymerases