Characterization of the pathway-specific positive transcriptional regulator for actinorhodin biosynthesis in Streptomyces coelicolor A3(2) as a DNA-binding protein

The ActII-ORF4 protein has been characterized as a DNA-binding protein that positively regulates the transcription of the actinorhodin biosynthetic ge nes. The target regions for the ActII-ORF4 protein were located within the act cluster. These regions, at high copy number, generate a nonproducer str ain by in vivo titration of the regulator. The mutant phenotype could be ma de to revert with extra copies of the wild-type actII-ORF4 gene but not wit h the actII-ORF4-177 mutant. His-tagged recombinant wild-type ActII-ORF4 an d mutant ActII-ORF4-177 proteins were purified from Escherichia coli cultur es; both showed specific DNA-binding activity for the actVI-ORF1-ORFA and a ctIII-actI intergenic regions. DNase I footprinting assays clearly located the DNA-binding sites within the -35 regions of the corresponding promoters , showing the consensus sequence 5'-TCGAG-3'. Although both gene products ( wild-type and mutant ActII-ORF4) showed DNA-binding activity, only the wild -type gene was capable of activating transcription of the act genes; thus, two basic functions can be differentiated within the regulatory protein: a specific DNA-binding activity and a transcriptional activation of the act b iosynthetic genes.