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首页> 外文期刊>Journal of bacteriology >The DnrN protein of Streptomyces peucetius, a pseudo-response regulator, is a DNA-binding protein involved in the regulation of daunorubicin biosynthesis.
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The DnrN protein of Streptomyces peucetius, a pseudo-response regulator, is a DNA-binding protein involved in the regulation of daunorubicin biosynthesis.

机译：伪链霉菌调节剂链霉菌的DnrN蛋白是一种涉及柔红霉素生物合成调控的DNA结合蛋白。

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摘要

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.
机译：DnrN是dnrI基因转录所必需的蛋白质，而dnrI基因又激活了Peuceomyces peucetius中柔红霉素生物合成基因的转录，在大肠杆菌和S. peucetius中过量生产。来自大肠杆菌的无细胞提取物用于进行DNA结合测定。凝胶迁移率迁移分析的结果表明，DnrN以高亲和力（Kd = 50 nM）与dnrI启动子区域特异性结合。乙酰磷酸盐和ATP均不影响结合能力，并且野生型DnrN与缺乏应答调节蛋白假定的磷酸化位点（天冬氨酸55）的突变形式（D-55-> N）之间的结合没有差异。因此，DnrN的磷酸化显然不是DNA结合所必需的。 DNase I足迹分析表明在dnrI转录起点上游37至55 bp和62至100 bp的结合区。有趣的是，这些区域的序列包括连续重叠的三元组[5'-（A / T）GC，5'-（A / T）CG，5'-（A / T）C（A / T）]被证明是柔红霉素的优先结合位点（JB Chaires and JE Herrera，Biochemistry 29：6145-6153，1990）。这可以解释为什么柔红霉素似乎会抑制DnrN与dnrI启动子的结合，从而导致柔红霉素产生的反馈抑制。用带有His标签的DnrN抗血清进行Western印迹（免疫印迹）分析的结果表明，dnrN表达与柔红霉素产生同时发生，并且野生型S. Peucetius菌株中DnrN的最高含量为总蛋白的0.01％。由于在不产生柔红霉素的突变菌株中DnrN的水平降低，我们推测柔红霉素调节dnrN和dnrI的表达。

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