Interactions of NodD at the nod Box: NodD binds to two distinct sites on the same face of the helix and induces a bend in the DNA.
Interactions of NodD at the nod Box: NodD binds to two distinct sites on the same face of the helix and induces a bend in the DNA.
复制标题
DOI：
10.1006/jmbi.1993.1515
复制
发表时间：
1993-10
影响因子：
5.6
通讯作者：
R. F. Fisher;S. R. Long
中科院分区：
文献类型：
--
作者：
R. F. Fisher;S. R. Long
苜蓿根瘤菌 nodD 基因产物是苜蓿根瘤菌-苜蓿共生中根瘤形态发生早期所需基因（nod 基因）的正转录激活剂。 NodD 是 LysR 激活蛋白家族的成员，其调节活性部分是通过其与位于诱导型 nod 基因上游的称为 nod 盒的保守 DNA 序列结合来介导的。在这里，我们使用干扰足迹来识别 nodA、nodF 和 nodH nod 盒中的两个 NodD 结合位点。这两个结合位点位于 DNA 螺旋的同一面上，可以额外分开 10 bp，同时保留活性。通过系统地改变 DNA 螺旋上两个结合位点的定相，我们发现只有在螺旋同一侧包含两个位点的构建体才能被 NodD 识别，这通过迁移阻滞测定和 nod box-lacZ 融合体的体内激活来确定。此外，NodD 显然在与 nod box 结合时诱导 DNA 弯曲，如循环排列的 nod box 片段的迁移阻滞行为所示。
The Rhizobium meliloti nodD gene products are positive transcriptional activators of genes required for early stages of nodule morphogenesis in the R. meliloti-alfalfa symbiosis (nod genes). The regulatory activity of NodD, a member of the LysR family of activator proteins, is mediated in part through its binding to conserved DNA sequences termed nod boxes which lie upstream of the inducible nod genes. Here we use interference footprinting to identify two NodD binding sites in the nodA, nodF and nodH nod boxes. These two binding sites are located on the same face of the DNA helix and can be separated by an additional 10 bp with retention of activity. By systematic alteration of the phasing of the two binding sites on the DNA helix, we showed that only constructs which contain both sites on the same side of the helix are recognized by NodD as determined by migration retardation assay and by in vivo activation of nod box-lacZ fusions. Moreover, NodD apparently induces a bend in the DNA upon binding at the nod box as shown by migration retardation behavior of circularly permuted nod box fragments.