BPS2026 – Conformational switching in RNase E regulates RNA binding selectivity and BR-body phase separation

Cells across all domains of life use ribonucleoprotein condensates to organize RNA metabolism. In bacteria, these condensates, known as bacterial ribonucleoprotein bodies (BR-bodies), form in response to RNA and dissolve upon RNA degradation. Yet how BR-bodies selectively recognize RNA while avoiding nonspecific interactions with DNA or membranes remains poorly understood. Here, we investigate the role of the C-terminal domain (CTD) of Caulobacter crescentus ribonuclease E (RNase E), a key phase-separating component of BR-bodies. The CTD contains an intrinsically disordered region composed of alternating charged blocks: multivalent, arginine-rich “sticker” blocks that bind RNA and promote phase separation, interspersed with negatively charged “spacer” regions. Through cell-based assays and in vitro EMSA assays, we found that multivalent charged blocks are necessary to distinguish single-stranded RNA from both single-stranded and duplexed DNA, suggesting that multivalent 2’OH recognition is critical for RNA recognition over DNA. We also examined the role of the negatively charged spacer regions. We observed that a reduction in the negative charge of the spacers led to the accumulation of RNase E along the negatively charged membrane. This indicates that the negatively charged spacers play a role in preventing off-target binding to other negatively charged binding sites. To probe conformation changes within RNase E’s disordered region with donors and acceptors labeled at different positions. We found that RNase E adopts a closed conformation in the absence of RNA, potentially restricting access to the arginine-rich blocks. In contrast, RNA or phase-separation inducers (e.g., PEG(8000) or MgCl 2 ) promote a more open conformation. Together, our results support a model in which RNase E’s multi-block intrinsically disordered region acts as a conformational switch that restricts nonspecific interactions with DNA or membranes while enabling selective recognition of RNA, thereby driving BR-body assembly.