title: DNA-binding specificity of AdpA, a transcriptional activator in the A-factor regulatory cascade in Streptomyces griseus.
abstract: AdpA, belonging to the AraC/XylS family, is the key transcriptional activator for a number of genes of various functions in the A-factor regulatory cascade in Streptomyces griseus. It consists of a ThiJ/PfpI/DJ-1-like dimerization domain at its N-terminal portion and a DNA-binding domain with two helix-turn-helix motifs at its C-terminal portion, representing a large subgroup of the AraC/XylS family. Uracil interference assay and missing T and GA interference assays on several AdpA binding sites, followed by gel mobility shift assays on systematically mutated binding sites, revealed a consensus AdpA-binding sequence, 5'-TGGCSNGWWY-3' (S: G or C; W: A or T; Y: T or C; N: any nucleotide). A dimer of AdpA bound a site including the two consensus sequences, with a space of 13-14 bp, as an inverted repeat (type I) at various positions, for example more than 200 bp upstream (-200) and 25 bp downstream (+25) from the transcriptional start point of the target gene. In addition, AdpA also bound a site including the consensus sequence in a single copy (type II) at positions, in most cases, from -40 to -50 and from -50 to -60. For transcriptional activation, some genes required simultaneous binding of a dimer of AdpA to type I and II sites, but others required only a single type I or type II site. AdpA bound mutated type I sites with various distances between the two consensus sequences with significant affinities, although the optimal distances for AdpA to bind were 13-14 bp and 2 bp. The DNA-binding domain is therefore connected to the ThiJ/PfpI/DJ-1-like dimerization domain with a flexible linker. The DNA-binding specificity of AdpA in conjunction with that of other AraC/XylS family members is discussed.
authors.affiliation: Department of Biotechnology, Graduate School of Agriculture and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan.
authors.forename: Haruka
authors.initials: H
authors.lastname: Yamazaki
authors.forename: Ayami
authors.initials: A
authors.lastname: Tomono
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Ohnishi
authors.forename: Sueharu
authors.initials: S
authors.lastname: Horinouchi
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D016384:Consensus Sequence; D004269:DNA, Bacterial / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism*; D019281:Dimerization; D015964:Gene Expression Regulation, Bacterial; D005797:Genes, araC; D019077:Helix-Turn-Helix Motifs; D008969:Molecular Sequence Data; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013305:Streptomyces griseus / Q000235:genetics* / Q000378:metabolism*; D013379:Substrate Specificity; D015534:Trans-Activators / Q000235:genetics* / Q000378:metabolism* / Q000502:physiology; D015533:Transcriptional Activation
chemical_list: C401645:AdpA protein, Streptomyces griseus; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D015534:Trans-Activators; C078720:XylS protein, Pseudomonas putida