The CTXφ repressor RstR binds DNA cooperatively to form tetrameric repressor-operator complexes
The CTXφ repressor RstR binds DNA cooperatively to form tetrameric repressor-operator complexes
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DOI：
10.1074/jbc.m311109200
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发表时间：
2004-01-23
影响因子：
4.8
通讯作者：
Waldor, MK
中科院分区：
文献类型：
--
作者：
Kimsey, HH;Waldor, MK
CTXphi 是一种丝状噬菌体，编码霍乱毒素并整合到霍乱弧菌基因组中形成稳定的溶原菌。在 CTXphi 溶原菌中，源自 rstA 噬菌体启动子的基因表达被噬菌体编码的阻遏物 RstR 抑制。 RstR 的 N 端区域包含一个螺旋-转角-螺旋 DNA 结合元件，类似于噬菌体阻遏物 cI/Cro 家族的螺旋-转角-螺旋，而短的 C 端区域与 cI 阻遏物的寡聚结构域无关。纯化的带有 His 标签的 RstR 与 rstA 启动子区域中的三个延伸的 50 bp 操纵子位点结合。每个 RstR 足迹都表现出 DNase I 可访问区域的特征性交错模式，表明 RstR 以二聚体的二聚体形式结合 DNA。在凝胶渗透色谱和交联实验中，RstR 寡聚形成二聚体和四聚体。通过使用 RstR 和 RstR 的延长活性变体的混合物进行迁移率变化实验，显示当与操纵子 DNA 结合时，RstR 是四聚体。 RstR 与高亲和力 O1 位点的结合适合操纵子结合的协作模型，其中两个 RstR 二聚体结合形成四聚体 RstR-操纵子复合物。在迁移率变动分析中未观察到 RstR 二聚体与 O1 操纵基因 DNA 左半部分或右半部分的结合。这些观察结果支持一个模型，其中相邻 RstR 二聚体之间的蛋白质-蛋白质接触有助于强操纵子结合。
CTXphi is a filamentous bacteriophage that encodes cholera toxin and integrates into the Vibrio cholerae genome to form stable lysogens. In CTXphi lysogens, gene expression originating from the rstA phage promoter is repressed by the phage-encoded repressor RstR. The N-terminal region of RstR contains a helix-turn-helix DNA-binding element similar to the helix-turn-helix of the cI/Cro family of phage repressors, whereas the short C-terminal region is unrelated to the oligomerization domain of cI repressor. Purified His-tagged RstR bound to three extended 50-bp operator sites in the rstA promoter region. Each of the RstR footprints exhibited a characteristic staggered pattern of DNase I-accessible regions that suggested RstR binds DNA as a dimer-of-dimers. In gel permeation chromatography and crosslinking experiments, RstR oligomerized to form dimers and tetramers. RstR was shown to be tetrameric when bound to operator DNA by performing mobility shift experiments with mixtures of RstR and a lengthened active variant of RstR. Binding of RstR to the high affinity O1 site could be fit to a cooperative model of operator binding in which two RstR dimers associate to form tetrameric RstR-operator complexes. The binding of RstR dimers to the left or right halves of O1 operator DNA was not observed in mobility shift assays. These observations support a model in which protein-protein contacts between neighboring RstR dimers contribute to strong operator binding.