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, HSC 4H21, McMaster University, 1200 Main Street West, Hamilton, Ontario, Canada.
Journal of Bacteriology (Impact Factor: 2.81). 10/2007; 189(18):6655-64. DOI: 10.1128/JB.00717-07
Source: PubMed


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, Oact (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 Oact. 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.

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Available from: Kapil Tahlan, Feb 04, 2014
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    • "In a previous study, we designed a TetR-based biosensor that produced luminescence upon addition of tetracycline derivatives [8]. The goals of the work described here were to improve the sensitivity of this system and to then exploit it to detect the activities of tetracycline modifying enzymes. "
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