Investigation of transcription repression and small-molecule responsiveness by TetR-like transcription factors using a heterologous Escherichia coli-based assay
Investigation of transcription repression and small-molecule responsiveness by TetR-like transcription factors using a heterologous Escherichia coli-based assay
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DOI：
10.1128/jb.00717-07
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发表时间：
2007-09-01
影响因子：
3.2
通讯作者：
Nodwell, Justin
中科院分区：
文献类型：
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作者：
Ahn, Sang Kyun;Tahlan, Kapil;Nodwell, Justin
SCO 7222蛋白和ActR是天蓝色链霉菌基因组中编码的150个TetR样转录因子中的两个。使用生物发光作为读数，我们已经开发出基于大肠杆菌的生物传感器，准确地报告这些蛋白质的调节活性，并用它来研究它们与DNA和小分子配体的相互作用。我们发现SCO 7222蛋白和ActR通过与启动子中的操纵子序列相互作用，分别抑制其推定的靶基因SCO 7223和actII-ORF 2（actA）的表达。这两种蛋白识别的操纵子是相关的，因此O-1223（SCO 7223的操纵子）可以被SCO 7222蛋白和ActR以相似的亲和力结合。相反，O-act（actII-ORF 2的操纵子）与ActR紧密结合，与SCO 7222蛋白结合较弱。我们证明了这些蛋白质的配体特异性，通过显示，而TetR（而不是ActR或SCO 7222蛋白）与四环素类药物相互作用，ActR（而不是TetR或SCO 7222蛋白）与放线菌紫素和相关分子相互作用。通过操纵子靶向突变，我们发现O-7223中至少需要两个核苷酸的变化才能破坏其与SCO 7222蛋白的相互作用，而ActR对O-act上的变化更敏感。最重要的是，我们发现每种蛋白质与野生型和突变型操纵子序列在体内和体外的相互作用完全相关。我们的数据表明E.这种类型的基于大肠杆菌的生物传感器应该广泛适用于TetR样转录因子。
The SCO7222 protein and ActR are two of similar to 150 TetR-like transcription factors encoded in the Streptomyces coelicolor genome. Using bioluminescence as a readout, we have developed Escherichia coli-based biosensors that accurately report the regulatory activity of these proteins and used it to investigate their interactions with DNA and small-molecule ligands. We found that the SCO7222 protein and ActR repress the expression of their putative target genes, SCO7223 and actII-ORF2 (actA), respectively, by interacting with operator sequence in the promoters. The operators recognized by the two proteins are related such that O-1223 (an operator for SCO7223) could be bound by both the SCO7222 protein and ActR with similar affinities. In contrast, O-act (an operator for actII-ORF2) was bound tightly by ActR and more weakly by the SCO7222 protein. We demonstrated ligand specificity of these proteins by showing that while TetR (but not ActR or the SCO7222 protein) interacts with tetracyclines, ActR (but not TetR or the SCO7222 protein) interacts with actinorhodin and related molecules. Through operator-targeted mutagenesis, we found that at least two nucleotide changes in O-7223 were required to disrupt its interaction with SCO7222 protein, while ActR was more sensitive to changes on O-act. Most importantly, we found that the interaction of each protein with wild-type and mutant operator sequences in vivo and in vitro correlated perfectly. Our data suggest that E. coli-based biosensors of this type should be broadly applicable to TetR-like transcription factors.