The DnrN protein of Streptomyces peucetius, a pseudo-response regulator, is a DNA-binding protein involved in the regulation of daunorubicin biosynthesis.
DnrN, a protein essential for the transcription of the dnrI gene, which in turn activates transcription of the daunorubicin biosynthesis genes in Streptomyces peucetius, was overproduced in Escherichia coli and S. peucetius. The cell-free extract from E. coli was used to conduct DNA-binding assays. The results of gel mobility shift analysis showed that DnrN binds specifically to the dnrI promoter region with a high affinity (Kd = 50 nM). Neither acetyl phosphate nor ATP affected the binding ability, and there was no difference in binding between wild-type DnrN and a mutant form (D-55-->N) lacking the putative phosphorylation site (aspartate 55) of a response regulator protein. Therefore, phosphorylation of DnrN apparently is not necessary for DNA binding. DNase I footprinting analysis indicated binding regions at 37 to 55 bp and 62 to 100 bp upstream of the transcriptional start point of dnrI. Interestingly, the sequence of these regions includes consecutive overlapping triplets [5'-(A/T)GC, 5'-(A/T)CG, 5'-(A/T)C(A/T)] that have been shown to be the preferential binding site of daunorubicin (J. B. Chaires and J. E. Herrera, Biochemistry 29:6145-6153, 1990). This may explain why daunorubicin appeared to inhibit the binding of DnrN to the dnrI promoter, which could result in feedback repression of daunorubicin production. The results of Western blotting (immunoblotting) analysis with His-tagged DnrN antiserum showed that dnrN expression is coincident with daunorubicin production and that the maximum level of DnrN is 0.01% of total protein in the wild-type S. peucetius strain. Since the level of DnrN was lowered in mutant strains that do not produce daunorubicin, we speculate that dnrN and dnrI expression are regulated by daunorubicin.
Furuya
K
K
School of Pharmacy, University of Wisconsin, Madison 53706, USA.
Hutchinson
C R
CR
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins / Q000378:metabolism*; D003630:Daunorubicin / Q000737:chemistry / Q000378:metabolism*; D003850:Deoxyribonuclease I / Q000378:metabolism; D015870:Gene Expression; D008969:Molecular Sequence Data; D010755:Organophosphates / Q000378:metabolism; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D013302:Streptomyces / Q000378:metabolism*; D014157:Transcription Factors
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; C092188:DnrN protein, Streptomyces peucetius; D010755:Organophosphates; D014157:Transcription Factors; C011632:acetyl phosphate; D003850:Deoxyribonuclease I; D003630:Daunorubicin