The Escherichia coli RelB antitoxin C-terminus is essential for RelE toxin suppression and transcriptional repression

Julia Tanquary; Ian J Pavelich; Marcin Grabowicz; Christine M Dunham

doi:10.1016/j.jbc.2025.110389

The Escherichia coli RelB antitoxin C-terminus is essential for RelE toxin suppression and transcriptional repression

J Biol Chem. 2025 Jun 18:110389. doi: 10.1016/j.jbc.2025.110389. Online ahead of print.

Authors

Julia Tanquary¹, Ian J Pavelich², Marcin Grabowicz³, Christine M Dunham⁴

Affiliations

¹ Department of Chemistry, Emory University, Atlanta, GA 30322, USA; Graduate program in Biochemistry, Cell and Developmental Biology, Graduate Division of Biological and Biomedical Sciences, Emory University, Atlanta, GA 30322, USA.
² Department of Chemistry, Emory University, Atlanta, GA 30322, USA.
³ Emory Antibiotic Resistance Center, Emory University, Atlanta, GA 30322, USA; Department of Microbiology & Immunology, Emory University School of Medicine, Atlanta, GA 30322, USA; Division of Infectious Diseases, Department of Medicine, Emory University School of Medicine, Atlanta, GA 30322, USA.
⁴ Department of Chemistry, Emory University, Atlanta, GA 30322, USA; Emory Antibiotic Resistance Center, Emory University, Atlanta, GA 30322, USA.

PMID: 40541805
DOI: 10.1016/j.jbc.2025.110389

Abstract

Bacterial type II toxin-antitoxin (TA) systems exhibit high specificity within each pair to ensure precise recognition of the toxin by its cognate antitoxin to inhibit toxicity of the free toxin. Despite high structural similarity among some TAs, crosstalk between non-cognate TA pairs is rare. To determine how the E. coli RelB antitoxin suppresses its cognate RelE toxin, we engineered C-terminal truncations of RelB and tested their functional effects on toxin suppression in E. coli. We find that removal of the long C-terminal α3 and connecting loop 4 (L4) of RelB prevents RelE suppression. Quantitative binding assays of RelE and RelB variants support a reduced affinity upon removal of the RelB C-terminus. Disrupting these interactions between RelB and RelE also led to a significant decrease in transcriptional repression at the relO operator, underscoring the requirement for RelE binding to RelB for optimal repression at DNA repressor elements. Comparison to other structurally homologous TA systems, such as E. coli DinJ-YafQ, reveals key differences in the molecular mechanisms of both toxin suppression and DNA repressor activity highlighting the diversity in TA regulation and function.

Keywords: DNA-binding proteins; protein-protein interactions; toxin-antitoxin systems; transcriptional repression.