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DNA‐binding specificity of AdpA, a transcriptional activator in the A‐factor regulatory cascade in Streptomyces griseus
Author(s) -
Yamazaki Haruka,
Tomono Ayami,
Ohnishi Yasuo,
Horinouchi Sueharu
Publication year - 2004
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1111/j.1365-2958.2004.04153.x
Subject(s) - biology , streptomyces griseus , gene , consensus sequence , binding site , promoter , genetics , transcription factor , activator (genetics) , dna , dna binding site , microbiology and biotechnology , peptide sequence , streptomyces , gene expression , bacteria
Summary 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.