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 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 actII-ORF4 gene but not with the actII-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 actVI-ORF1-ORFA and actIII-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 biosynthetic genes.
Arias
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Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas, Campus Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain.
Fernández-Moreno
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MA
Malpartida
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D000880:Anthraquinones / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D018983:DNA Footprinting; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000378:metabolism; D017422:Sequence Analysis, DNA; D013302:Streptomyces / Q000235:genetics / Q000378:metabolism*; D015534:Trans-Activators / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic; D000095622:Benzoisochromanequinones
D000880:Anthraquinones; D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; D015534:Trans-Activators; C013390:actinorhodin; D000095622:Benzoisochromanequinones