Dynamic partitioning shapes the in vivo organization of the E. coli RNA degradosome

Macromolecular scaffolds are often viewed as fully assembled molecular machines with fixed stoichiometry. Here, we show that the Escherichia coli RNA degradosome follows a more dynamic organizational principle. Using live-cell single-molecule imaging, we quantified the spatial distribution, diffusion, and stoichiometry of RNase E and its accessory factors RhlB, PNPase, and enolase in vivo. The membrane-associated stoichiometry of RhlB and PNPase was broadly consistent with canonical expectation, whereas enolase was underrepresented in the membrane-bound degradosome. In contrast to the near-complete membrane association of RhlB, PNPase partitioned substantially between membrane-associated and cytoplasmic pools, and this partitioning shifted with RNA availability and growth condition. Using lacZ reporters with different translation initiation strengths, we further show that RhlB and PNPase preferentially promote degradation of weakly translated transcripts, whereas strongly translated transcripts are largely insensitive to their loss. Together, these results support a model in which the E. coli RNA degradosome is a membrane-anchored but dynamically assembled complex, with accessory factors contributing differently across physiological states and RNA substrate classes.