G-Quadruplex structures within the hfq gene regulate RNA–protein interactions in Acinetobacter baumannii

Abstract

G-quadruplexes (G4s) are non-canonical nucleic acid structures with emerging regulatory significance in bacterial gene expression. While extensively studied in eukaryotes, the roles of G4s especially two-tetrad (2G) G4s in prokaryotic systems remain greatly underexplored. In this study, we identified and characterized multiple 2G G4-forming motifs within the hfq gene of Acinetobacter baumannii, a clinically significant and highly resilient pathogen. The RNA chaperone Hfq protein plays a central role in post-transcriptional gene regulation in this organism. Using a combination of in silico prediction and biophysical techniques (NMR, CD spectroscopy, EMSA, fluorescence titration, and ITC), we determined the folding and topology of these motifs into stable G4 structures, particularly in RNA. These G4s showed high-affinity binding with BRACO-19, a known G4 ligand, and preferential interaction with full-length Hfq protein compared to its C-terminally truncated variant, underscoring the role of the glycine-rich C-terminal domain in RNA recognition. Furthermore, BRACO-19-mediated stabilization of these G4 structures resulted in significant downregulation of hfq transcript variants, especially in the glycine-rich region. Collectively, this work uncovers a novel regulatory axis involving G-quadruplexes and Hfq protein in A. baumannii, highlighting G4-Hfq interactions as potential antimicrobial targets and offering a scaffold for the broader exploration of RNA-based regulation in this pathogenic bacterium. © 2025 Elsevier Inc.