Present address: Division of Molecular Neurobiology, Institute of Medical Sciences, The University of Tokyo, Minato-ku, Tokyo 108-8639, Japan.
DNA-binding specificity of AdpA, a transcriptional activator in the A-factor regulatory cascade in Streptomyces griseus
Article first published online: 10 JUN 2004
Volume 53, Issue 2, pages 555–572, July 2004
How to Cite
Yamazaki, H., Tomono, A., Ohnishi, Y. and Horinouchi, S. (2004), DNA-binding specificity of AdpA, a transcriptional activator in the A-factor regulatory cascade in Streptomyces griseus. Molecular Microbiology, 53: 555–572. doi: 10.1111/j.1365-2958.2004.04153.x
- Issue published online: 10 JUN 2004
- Article first published online: 10 JUN 2004
- Accepted 30 March, 2004.
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.