The SCO7222 protein and ActR are two of approximately 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(7223) (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.

译文

SCO7222蛋白和ActR是coelicolor链霉菌基因组中编码的大约150个TetR样转录因子中的两个。使用生物发光作为读数,我们开发了基于大肠杆菌的生物传感器,可准确报告这些蛋白质的调节活性,并将其用于研究它们与DNA和小分子配体的相互作用。我们发现,SCO7222蛋白和ActR通过与启动子中的操纵子序列相互作用,分别抑制了其假定的靶基因SCO7223和actII-ORF2 (actA) 的表达。由两种蛋白质识别的算子相关,使得O(7223) (SCO7223的算子) 可以被SCO7222蛋白和ActR结合,具有相似的亲和力。相反,O(act) (actII-ORF2的操作员) 被ActR紧密结合,而被SCO7222蛋白更弱地结合。我们通过显示TetR (但不是ActR或SCO7222蛋白) 与四环素相互作用,ActR (但不是TetR或SCO7222蛋白) 与放线菌素和相关分子相互作用,证明了这些蛋白的配体特异性。通过操作员靶向诱变,我们发现至少需要两个核苷酸变化O(7223) 来破坏其与SCO7222蛋白的相互作用,而ActR对O(act) 的变化更敏感。最重要的是,我们发现每种蛋白质在体内和体外与野生型和突变算子序列的相互作用完全相关。我们的数据表明,这种类型的基于大肠杆菌的生物传感器应广泛适用于TetR样转录因子。

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