The tetracycline repressor family transcriptional regulators (TFRs) are homodimeric DNA-binding proteins that generally act as transcriptional repressors. Their DNA-binding activity is allosterically inactivated by the binding of small-molecule ligands. TFRs constitute the third most frequently occurring transcriptional regulator family found in bacteria with more than 10,000 representatives in the nonredundant protein database. In addition, more than 100 unique TFR structures have been solved by X-ray crystallography. In this study, we have used computational and experimental approaches to reveal the variations and conservation present within TFRs. Although TFR structures are very diverse, we were able to identify a conserved central triangle in their ligand-binding domains that forms the foundation of the structure and the framework for the ligand-binding cavity. While the sequences of DNA-binding domains of TFRs are highly conserved across the whole family, the sequences of their ligand-binding domains are so diverse that pairwise sequence similarity is often undetectable. Nevertheless, by analyzing subfamilies of TFRs, we were able to identify distinct regions of conservation in ligand-binding domains that may be important for allostery. To aid in large-scale analyses of TFR function, we have developed a simple and reliable computational approach to predict TFR operator sequences, a temperature melt-based assay to measure DNA binding, and a generic ligand-binding assay that will likely be applicable to most TFRs. Finally, our analysis of TFR structures highlights their flexibility and provides insight into a conserved allosteric mechanism for this family.
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