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

Area of Science:

• Bacterial gene regulation
• Nucleic acid structure
• Antimicrobial research

Background:

• G-quadruplexes (G4s) are non-canonical nucleic acid structures with emerging regulatory roles.
• Their function in prokaryotes, especially two-tetrad (2G) G4s, is underexplored.
• Acinetobacter baumannii is a resilient, clinically significant pathogen where Hfq protein regulates gene expression.

Purpose of the Study:

• To identify and characterize 2G G4-forming motifs in the hfq gene of A. baumannii.
• To investigate the interaction between these G4s and the Hfq protein.
• To explore the potential of G4-Hfq interactions as antimicrobial targets.

Main Methods:

• In silico prediction of G4 motifs.
• Biophysical techniques including NMR, CD spectroscopy, EMSA, fluorescence titration, and ITC.
• Analysis of BRACO-19 mediated G4 stabilization and its effect on hfq transcript levels.

Main Results:

• Multiple 2G G4 motifs were identified and characterized in the A. baumannii hfq gene, forming stable structures.
• These G4s exhibit high-affinity binding to the G4 ligand BRACO-19.
• G4s preferentially interact with full-length Hfq protein, indicating the C-terminal domain's importance in RNA recognition.
• BRACO-19 stabilized G4s led to significant downregulation of hfq transcript variants.

Conclusions:

• A novel regulatory axis involving G-quadruplexes and Hfq protein in A. baumannii is uncovered.
• G4-Hfq interactions represent potential antimicrobial targets.
• This work provides a foundation for exploring RNA-based regulation in pathogenic bacteria.