Characterization of the Pathway-Specific Positive Transcriptional Regulator for Actinorhodin Biosynthesis in Streptomyces coelicolor A3(2) as a DNA-Binding Protein

Abstract

ABSTRACT The ActII-ORF4 protein has been characterized as a DNA-binding protein that positively regulates the transcription of the actinorhodin biosynthetic genes. The target regions for the ActII-ORF4 protein were located within the act cluster. These regions, at high copy number, generate a nonproducer strain by in vivo titration of the regulator. The mutant phenotype could be made to revert with extra copies of the wild-type act II-ORF4 gene but not with the act II-ORF4-177 mutant. His-tagged recombinant wild-type ActII-ORF4 and mutant ActII-ORF4-177 proteins were purified from Escherichia coli cultures; both showed specific DNA-binding activity for the act VI-ORF1–ORFA and act III- act I 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 biosynthetic genes.