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This study reveals how the bacterial protein CopY regulates copper levels through an asymmetric dual-operator mechanism. Using biophysical techniques including circular dichroism and electron paramagnetic resonance, researchers found that CopY binds two DNA operator sites with different affinities—a high-affinity proximal site that anchors the protein and a lower-affinity distal site that remodels the promoter. The metal binding domain remains flexible even when bound to DNA, which may allow rapid copper sensing and release of repression when intracellular copper rises.
Why it matters
Understanding copper homeostasis mechanisms is important for developing antibacterial strategies and bioengineering applications, as copper regulation is essential for bacterial survival. The discovery of asymmetric operator binding providing complementary regulatory functions offers insights into how bacteria fine-tune gene expression in response to metal availability.
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⚠️ Preprint – Noch nicht peer-reviewed
Dieser Artikel wurde noch nicht von unabhängigen Experten begutachtet. Die Ergebnisse sind vorläufig und sollten mit Vorsicht interpretiert werden.
Copper is essential yet potentially toxic, requiring tight regulation of intracellular concentration. The copper-responsive transcriptional repressor CopY is unusual among bacterial metalloregulators because it occupies two operator sites within the cop promoter. Although two CopY dimers bind these operators independently, the mechanistic significance of this dual-operator architecture remains unclear. Here, we combine biochemical assays, circular dichroism (CD), and electron paramagnetic resonance (EPR) distance measurements to define the molecular basis of CopY-mediated repression. CopY binds the proximal operator with higher affinity than the distal operator. CD measurements further show that DNA binding increases CopY thermal stability, with greater stabilization by the proximal operator. Conversely, DNA-based EPR measurements reveal comparable CopY-induced compaction of both operators. Thus, protein stabilization and DNA deformation represent distinct components of repression. Moreover, site-directed spin labeling and EPR demonstrate that the metal binding domain is conformationally heterogeneous and highly flexible. DNA binding has only a marginal effect on this flexibility. Thus, the metal site is not a rigid pocket but a dynamic one, even in the DNA-bound repressor, a property that may facilitate Cu(I) capture and hence derepression once copper levels rise. We propose that the proximal operator serves as a high-affinity anchoring site, whereas the distal operator primarily promotes promoter remodeling, and that the plasticity of the metal binding domain is retained in the DNA-bound repressor. Together, these findings support a "transcriptional duet" model in which two CopY dimers bound to non-equivalent operators make distinct and complementary contributions to repression.
Source: CopY as a Transcriptional Duet Orchestrating Copper Homeostasis via Asymmetry-Dependent DNA Affinity