Structural basis for higher-order DNA binding by a bacterial transcriptional regulator
Frederik Oskar Graversgaard Henriksen, Lan Bich Van, Ditlev Egeskov Brodersen, RagnhildBager Skjerning
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.
Introduction
Regulation of transcription is a fundamental mechanism in bacteria that allows adaptation to constantly changing environments, and RNA polymerase (RNAP) plays a central part in this. The core RNAP enzyme has a ββ′α2 subunit composition [1] but in order to form a holoenzyme and initiate transcription at a particular promoter, the core enzyme needs to interact with a σ subunit [2,3].
Materials and method
Bacterial strains and plasmids
Bacterial strains and plasmids used in this study are listed in S2 Table, and DNA oligonucleotides in S3 Table. Detailed descriptions of plasmid construction are found in S1 Text. For site-directed mutagenesis, the Q5 Site-Directed Mutagenesis Kit from NEB was used according to the protocol and mutagenesis primers were designed using NEBaseChanger.
Results
The P. putida xre-res locus is transcriptionally auto-regulated
The presence of a DNA-binding HTH domain in XrePp suggested that the antitoxin and/or the Xre-RES TA complex could be involved in regulating transcription of the P. putida xre-res operon via direct promoter binding. To test this hypothesis, we analysed the xre-resPp promoter region (PXR) and identified three inverted repeats and one directional repeat, named Sequence 1 to 4 (S1-S4) (Figs 1A and S1A).
Discussion
In this paper, we present a detailed model for transcriptional autoregulation of the P. putida xre-res operon. We show that the heterohexameric Xre-RESPp protein complex efficiently represses transcription from the xre-res locus through direct binding to an imperfect inverted repeat region (S4) in the promoter region, and the structure of the Xre-RESPp complex bound to this DNA motif provides a detailed molecular view of how the promoter is specifically recognised.
Acknowledgments
The authors would like to thank Janni Nielsen and Prof. Daniel E. Otzen for help with SEC-MALS, Camilla G. Andersen for help with XRD data analysis, Rasmus Freund for scripts designed to plot the pLDDT and PAE files from the AlphaFold 3 webserver, and Dr. Kristoffer Winther for help with identification of the four promoter repeat sequences.
Citation: Henriksen FOG, Van LB, Brodersen DE, Skjerning R (2025) Structural basis for higher-order DNA binding by a bacterial transcriptional regulator. PLoS Genet 21(6): e1011749. https://doi.org/10.1371/journal.pgen.1011749
Editor: Kai Papenfort, Friedrich-Schiller-Universitat Jena, GERMANY
Received: February 19, 2025; Accepted: May 27, 2025; Published: June 27, 2025
Copyright: © 2025 Henriksen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the manuscript and its Supporting Information files.
Funding: This study was funded by the Independent Research Fund Denmark, grant no. 0135-00072B and by the Novo Nordisk Foundation, grants NNF18OC0030646 and NNF22OC0079855 to D.E.B. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
