Mapping the DNA-binding domain and target sequences of the Streptomyces peucetius daunorubicin biosynthesis regulatory protein, DnrI.
Streptomyces antibiotic regulatory proteins (SARPs) constitute a novel family of transcriptional activators that control the expression of several diverse anti-biotic biosynthetic gene clusters. The Streptomyces peucetius DnrI protein, one of only a handful of these proteins yet discovered, controls the biosynthesis of the polyketide antitumour antibiotics daunorubicin and doxorubicin. Recently, comparative analyses have revealed significant similarities among the predicted DNA-binding domains of the SARPs and the C-terminal DNA-binding domain of the OmpR family of regulatory proteins. Using the crystal structure of the OmpR-binding domain as a template, DnrI was mapped by truncation and site-directed mutagenesis. Several highly conserved residues within the N-terminus are crucial for DNA binding and protein function. Tandemly arranged heptameric imperfect repeat sequences are found within the -35 promoter regions of target genes. Substitutions for each nucleotide within the repeats of the dnrG-dpsABCD promoter were performed by site-directed mutagenesis. The mutant promoter fragments were found to have modified binding characteristics in gel mobility shift assays. The spacing between the repeat target sequences is also critical for successful occupation by DnrI and, therefore, competent transcriptional activation of the dnrG-dpsABCD operon.
Sheldon
Paul J
PJ
School of Pharmacy, University of Wisconsin, 425 N. Charter Street, Madison 53706, USA.
Busarow
Sara B
SB
Hutchinson
C Richard
CR
D001483:Base Sequence; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism*; D003630:Daunorubicin / Q000096:biosynthesis*; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015183:Restriction Mapping; D017384:Sequence Deletion; D013302:Streptomyces / Q000235:genetics* / Q000378:metabolism
D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D003630:Daunorubicin