Seven amino acids gate transcriptional activation by a minimal MarA Helix turn-helix DNA-binding domain
- 1Departament de Bioquimica i Biologia Molecular, Universitat Autonoma de Barcelona; 08193 Cerdanyola del Valles, Spain
Abstract
Prokaryotic transcription factors (TFs) lie at the core of antimicrobial resistance, controlling genes that let bacteria survive antibiotic exposure. The AraC/XylS family of TFs is defined by a ~99-residue DNA-binding domain composed of two helix-turn-helix (HTH) motifs. While this two-motif architecture is considered the minimal functional unit, the striking sequence and structural similarity between both HTH motifs raises the question of whether it evolved from a single ancestral HTH domain. Here, we designed two C-terminal truncations of MarA, comprising a single HTH motif, differing by seven-residues (IRSRKMT). Electrophoretic-mobility shift assays reveal that both constructs specifically bind the marbox sequence as reconstituted dimers, while size-exclusion chromatography shows they exist in a monomer-dimer equilibrium in solution. Despite retaining DNA-binding capacity, the truncations diverge functionally: while MarA64 (including IRSRKMT) activates transcription and confers regular erythromycin tolerance, MarA57 (lacking IRSRKMT) yields a transcriptionally inactive complex that suppresses reporter expression below baseline, suggesting competitive promoter occupancy. Molecular dynamics simulations and AlphaFold models suggest that the IRSRKMT extension forms an -helical element stabilizing a transcriptionally productive dimer interface. Conversely, its loss disrupts quaternary assembly, alters DNA bending, and misaligns RNA polymerase-contacting residues. Furthermore, free dimers explore non-productive conformations, suggesting that functional dimerization occurs upon DNA engagement. These findings establish that a single, correctly dimerized HTH domain is sufficient for both DNA binding and transcriptional activation, providing a structural rationale for short AraC/XylS-like proteins and offering a tuneable scaffold for synthetic biology and novel anti-virulence strategies.
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BibTeX
@misc{corbella2026seven,
title = {{Seven amino acids gate transcriptional activation by a minimal MarA Helix turn-helix DNA-binding domain}},
author = {M. Corbella and A. Serrano and R. Bello-Madruga and M. Torrent and I. Erill and J. M. A. Blair and E. Sancho-Vaello},
howpublished = {bioRxiv},
year = {2026},
month = sep,
doi = {10.64898/2026.09.23.753698},
url = {https://www.biorxiv.org/content/10.64898/2026.09.23.753698v1},
note = {CC BY},
}
RIS
TY - UNPB
TI - Seven amino acids gate transcriptional activation by a minimal MarA Helix turn-helix DNA-binding domain
AU - M. Corbella
AU - A. Serrano
AU - R. Bello-Madruga
AU - M. Torrent
AU - I. Erill
AU - J. M. A. Blair
AU - E. Sancho-Vaello
PY - 2026
DA - 2026/09/23
PB - bioRxiv
DO - 10.64898/2026.09.23.753698
UR - https://www.biorxiv.org/content/10.64898/2026.09.23.753698v1
AB - Prokaryotic transcription factors (TFs) lie at the core of antimicrobial resistance, controlling genes that let bacteria survive antibiotic exposure. The AraC/XylS family of TFs is defined by a ~99-residue DNA-binding domain composed of two helix-turn-helix (HTH) motifs. While this two-motif architecture is considered the minimal functional unit, the striking sequence and structural similarity between both HTH motifs raises the question of whether it evolved from a single ancestral HTH domain. Here, we designed two C-terminal truncations of MarA, comprising a single HTH motif, differing by seven-residues (IRSRKMT). Electrophoretic-mobility shift assays reveal that both constructs specifically bind the marbox sequence as reconstituted dimers, while size-exclusion chromatography shows they exist in a monomer-dimer equilibrium in solution. Despite retaining DNA-binding capacity, the truncations diverge functionally: while MarA64 (including IRSRKMT) activates transcription and confers regular erythromycin tolerance, MarA57 (lacking IRSRKMT) yields a transcriptionally inactive complex that suppresses reporter expression below baseline, suggesting competitive promoter occupancy. Molecular dynamics simulations and AlphaFold models suggest that the IRSRKMT extension forms an -helical element stabilizing a transcriptionally productive dimer interface. Conversely, its loss disrupts quaternary assembly, alters DNA bending, and misaligns RNA polymerase-contacting residues. Furthermore, free dimers explore non-productive conformations, suggesting that functional dimerization occurs upon DNA engagement. These findings establish that a single, correctly dimerized HTH domain is sufficient for both DNA binding and transcriptional activation, providing a structural rationale for short AraC/XylS-like proteins and offering a tuneable scaffold for synthetic biology and novel anti-virulence strategies.
ER -