Dual Role of Small Noncoding RNA and Hfq in Bacterial DNA Compaction: A New Perspective on Nucleoid Architecture.

The spatial organization of bacterial chromosomes is commonly attributed to nucleoid-associated proteins that compact DNA through bending and bridging interactions. Yet, the possible structural role of RNA in this process remains largely unexplored. Here we show that a small noncoding RNA (sRNA), DsrA, directly modulates DNA organization through its interaction with the amyloidogenic C-terminal region (CTR) of the nucleoid associated protein and RNA chaperone Hfq. Using nanofluidic confinement, we find that the Hfq-CTR strongly compacts λ-DNA, whereas addition of sRNA partially suppresses this condensation despite simultaneous association of DNA and RNA with the CTR peptide. Synchrotron radiation circular dichroism (SRCD) spectroscopy reveals that the CTR, in the presence of sRNA, induces a conformational rearrangement of AT-rich DNA consistent with altered base stacking and increased stiffness. Passive particle-tracking microrheology shows that DNA-CTR assemblies form heterogeneous viscoelastic gels. Addition of sRNA increases the elastic modulus and shortens the relaxation time, indicating a more connected yet dynamically responsive network. When interpreted together with the nanochannel measurements, these findings reveal a decoupling between local DNA compaction and bulk mechanical rigidity: sRNA reduces DNA collapse at the molecular scale while enhancing connectivity of the DNA-peptide network at the mesoscale. We propose that sRNAs can regulate nucleoid architecture not only through gene regulation but also by directly tuning DNA mechanics via protein-mediated cross-linking. These findings identify sRNA as an active structural component of bacterial chromosomal organization and reveal a multiscale mechanism by which RNA-protein interactions control the physical state of the bacterial genome.