Department of Genetic Engineering, School of Biotechnology, Madurai Kamaraj University, Madurai, Tamil Nadu, India.
Departments of Medicine, Physiology and Biophysics, University of California, Irvine, California, USA.
J Basic Microbiol. 2017 Oct;57(10):862-872. doi: 10.1002/jobm.201700162. Epub 2017 Jul 26.
The master regulator, DnrI of Streptomyces peucetius is a member of the family of transcriptional activator, Streptomyces antibiotic regulatory proteins (SARP), which controls the biosynthesis of antitumor anthracycline, daunorubicin (DNR) and doxorubicin (DXR). The binding of DnrI to the heptameric repeat sequence found within the -35 promoter region of biosynthetic gene, dpsE activates it. To combat the increased level of intracellular DNR, the cell has developed self resistance mechanism mediated by drrAB and drrC genes which are regulated by regulatory genes. We find that a drug non-producing mutant, ΔdpsA, showed sensitive phenotype in plate assay along with an increased level of dnrI transcript. Whereas the mutant grown in the presence of DNR showed a resistant phenotype with a six and eight folds increase in drrAB and drrC transcripts respectively. Computational studies followed by molecular docking showed that DnrI bound as a monomer to a slightly modified heptameric DNA motif, 5'-ACACGCA in drrA and 5'-ACAACCT in drrC which was also proved by electrophoretic mobility shift assay. These findings confirm that DnrI belongs to winged helix-turn-helix DNA-binding protein with Tetratricopeptide Repeat domain. The transcriptional regulator DnrI binds to the resistance genes at specific sites but they are activated only when an increased load of intracellular DNR is sensed.
主调控因子 DnrI 是链霉菌属抗生素调控蛋白 (SARP) 家族的成员，它控制抗肿瘤蒽环类抗生素柔红霉素 (DNR) 和多柔比星 (DXR) 的生物合成。DnrI 与生物合成基因 dpsE 的 -35 启动子区域内发现的七聚体重复序列结合，从而激活它。为了对抗细胞内 DNR 水平的升高，细胞已经开发出自我抵抗机制，该机制由 drrAB 和 drrC 基因介导，受调节基因调控。我们发现，一个不产药的突变体ΔdpsA 在平板测定中表现出敏感表型，同时 dnrI 转录本水平升高。而在 DNR 存在的情况下生长的突变体表现出抗性表型，drrAB 和 drrC 转录本分别增加了六倍和八倍。计算研究和分子对接表明，DnrI 作为单体与稍微修饰的七聚体 DNA 基序结合，在 drrA 中为 5'-ACACGCA，在 drrC 中为 5'-ACAACCT，这也通过电泳迁移率变动分析得到了证实。这些发现证实 DnrI 属于具有四肽重复结构域的翼螺旋-转角-螺旋 DNA 结合蛋白。转录调节因子 DnrI 与特定的抗性基因结合，但只有当细胞内 DNR 负荷增加时，它们才被激活。
