DNA footprinting and biophysical characterization of the controller protein C.AhdI suggests the basis of a genetic switch
DNA footprinting and biophysical characterization of the controller protein C.AhdI suggests the basis of a genetic switch
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DOI：
10.1093/nar/gkh975
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发表时间：
2004-01-01
影响因子：
14.9
通讯作者：
Kneale, GG
中科院分区：
文献类型：
--
作者：
Streeter, SD;Papapanagiotou, I;Kneale, GG
我们克隆并表达了AhdI限制修饰系统的ahdIC基因，并将所得控制器（C）蛋白纯化至均一。该蛋白质序列显示了在许多转录调节因子中发现的典型HTH基序。发现AhdI形成16.7 kDa的同源二聚体;沉降平衡实验表明二聚体在低浓度下解离成单体，解离常数为2.5 μ M。使用DNase I和Exo III足迹法来确定C.AhdI DNA结合位点，发现该位点与ahdIC操纵子上游30 bp相似。通过凝胶阻滞分析和表面等离子体共振判断，完整的同二聚体以5-6 nM的解离常数协同结合至含有C蛋白结合位点的35 bp DNA片段，尽管在实践中，对DNA的亲和力由蛋白质二聚化主导，因为单体对DNA的结合是可忽略的。的位置的C-运营商上游的ahdIC和ahdIR表明，C.AhdI可能作为一个积极的调节这两个基因的表达，并可以作为一个分子开关，是严重依赖于K-D的单体-二聚体平衡。此外，C.AhdI结合位点相对于C基因前假定的-35盒的结构和位置表明C.AhdI表达的自动调节的可能机制。
We have cloned and expressed the ahdIC gene of the AhdI restriction-modification system and have purified the resulting controller (C) protein to homogeneity. The protein sequence shows a HTH motif typical of that found in many transcriptional regulators. C.AhdI is found to form a homodimer of 16.7 kDa; sedimentation equilibrium experiments show that the dimer dissociates into monomers at low concentration, with a dissociation constant of 2.5 muM. DNase I and Exo III footprinting were used to determine the C.AhdI DNA-binding site, which is found similar to30 bp upstream of the ahdIC operon. The intact homodimer binds cooperatively to a 35 bp fragment of DNA containing the C-protein binding site with a dissociation constant of 5-6 nM, as judged both by gel retardation analysis and by surface plasmon resonance, although in practice the affinity for DNA is dominated by protein dimerization as DNA binding by the monomer is negligible. The location of the C-operator upstream of both ahdIC and ahdIR suggests that C.AhdI may act as a positive regulator of the expression of both genes, and could act as a molecular switch that is critically dependent on the K-d for the monomer-dimer equilibrium. Moreover, the structure and location of the C.AhdI binding site with respect to the putative -35 box preceding the C-gene suggests a possible mechanism for autoregulation of C.AhdI expression.