Electron Paramagnetic Resonance Spectroscopy Reveals Promoter Dependent Transcription Regulation by Copper Activated...

Metal responsive transcription factors are essential for bacterial metal homeostasis, allowing cells to regulate metal uptake, efflux, and detoxification in response to fluctuating metal ion levels. Among these, CueR, a member of the MerR family, is widely found in Gram-negative bacteria. While E. coli CueR has been extensively studied, revealing that it adopts multiple conformational states to regulate transcription, P. aeruginosa CueR (PACueR) remains less characterized, with no resolved structure despite regulating a broader set of genes. In this study, we applied electron paramagnetic resonance (EPR) spectroscopy combined with DNA spin-labeling to investigate the conformational states of PACueR bound to two different promoter sequences, copZ2 and mexPQ-opmE. We examined the effects of PACueR binding and copper addition, capturing the transcription initiation stage that represents an essential step in copper homeostasis regulation of P. aeruginosa. Our results reveal promoter-specific differences in PACueR DNA interactions, suggesting that while the core transcription initiation mechanism is conserved, variations in promoter affinity and length of dyad symmetry fine-tune transcription levels in response to copper. These findings highlight the value of EPR spectroscopy in probing metal-dependent transcription mechanisms and offer new insights into copper regulation in P. aeruginosa, a clinically important pathogen.