Structural basis for higher-order DNA binding by a bacterial transcriptional regulator

ABSTRACT Transcriptional regulation by binding of transcription factors to palindromic sequences in promoter regions is a fundamental process in bacteria. Some transcription factors have multiple dimeric DNA-binding domains, in principle enabling interaction with higher-order DNA structures; however, mechanistic and structural insights into this phenomenon remain limited. The Pseudomonas putida toxin-antitoxin (TA) system Xre-RES has an unusual 4:2 stoichiometry including two potential DNA-binding sites, compatible with a complex mechanism of transcriptional autoregulation. Here, we show that the Xre-RES complex interacts specifically with a palindromic DNA repeat in the promoter in a 1:1 molar ratio, leading to transcriptional repression. We determine the 2.7 Å crystal structure of the protein-DNA complex, revealing an unexpected asymmetry in the interaction and suggesting the presence of a secondary binding site, which is supported by structural prediction of the binding to the intact promoter region. Additionally, we show that the antitoxin can be partially dislodged from the Xre-RES complex, resulting in Xre monomers and a 2:2 Xre-RES complex, neither of which repress transcription. These findings highlight a dynamic, concentration-dependent model of transcriptional autoregulation, in which the Xre-RES complex transitions between a non-binding (2:2) and a DNA-binding (4:2) form. AUTHOR SUMMARY Bacteria regulate their gene expression to respond to environmental stress, to evade antibiotics, and to maintain population stability. In this study, we investigate how the xre-res toxin-antitoxin system from Pseudomonas putida controls its own expression. Using a combination of microbiology, structural biology, biophysical assays, and computational modelling, we discover how the Xre-RES protein complex represses its own transcription through direct binding to a specific DNA element in the promoter region. We show structurally that the Xre-RES complex adopts a unique 4:2 stoichiometry and binds DNA in an unusual asymmetrical manner. Moreover, the complex was found to shift between two different forms: one that binds DNA and represses transcription, and one that does not. We further demonstrate that this shift is dynamic and depends on the relative concentration of Xre antitoxin. Our findings provide new insight into how bacteria fine-tune gene expression and offers a model of transcriptional control based on protein stoichiometry and structural asymmetry.