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Investigation of Transcription Repression and Small-Molecule Responsiveness by TetR-Like Transcription Factors Using a Heterologous Escherichia coli-Based Assay

Department of Biochemistry and Biomedical Sciences, McMaster University, 1200 Main St. W., Hamilton, Ontario, Canada L8N 3Z5,¹ Department of Medical Genetics and Microbiology, University of Toronto, 1 King's College Circle, Toronto, Ontario, Canada M5S 1A8²

Received 7 May 2007/ Accepted 10 July 2007

The SCO7222 protein and ActR are two of 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 O₇₂₂₃ (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 O₇₂₂₃ 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.

Published ahead of print on 20 July 2007.