The abundant class of bacterial Hfq-associated small regulatory RNAs (sRNAs) parallels animal microRNAs in their ability to control multiple genes at the posttranscriptional level by short and imperfect base pairing. In contrast to the universal length and seed pairing mechanism of microRNAs, the sRNAs are heterogeneous in size and structure, and how they regulate multiple targets is not well understood. This paper provides evidence that a 5′ located sRNA domain is a critical element for the control of a large posttranscriptional regulon. We show that the conserved 5′ end of RybB sRNA recognizes multiple mRNAs of Salmonella outer membrane proteins by ≥7-bp Watson–Crick pairing. When fused to an unrelated sRNA, the 5′ domain is sufficient to guide target mRNA degradation and maintain σ ^E -dependent envelope homeostasis. RybB sites in mRNAs are often conserved and flanked by 3′ adenosine. They are found in a wide sequence window ranging from the upstream untranslated region to the deep coding sequence, indicating that some targets might be repressed at the level of translation, whereas others are repressed primarily by mRNA destabilization. Autonomous 5′ domains seem more common in sRNAs than appreciated and might improve the design of synthetic RNA regulators.

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RNase E, which is the central component of the multienzyme RNA degradosome, serves as a scaffold for interaction with other enzymes involved in mRNA degradation including the DEAD-box RNA helicase RhlB. Epifluorescence microscopy under live cell conditions shows that RNase E and RhlB are membrane associated, but neither protein forms cytoskeletal-like structures as reported earlier by Taghbalout and Rothfield. We show that association of RhlB with the membrane depends on a direct protein interaction with RNase E, which is anchored to the inner cytoplasmic membrane through an MTS (Membrane Targeting Sequence). Molecular dynamics simulations show that the MTS interacts with the phospholipid bilayer by forming a stabilized amphipathic α-helix with the helical axis oriented parallel to the plane of the bilayer and hydrophobic side chains buried deep in the acyl core of the membrane. Based on the molecular dynamics simulations, we propose that the MTS freely diffuses in the membrane by a novel mechanism in which a large number of weak contacts are rapidly broken and reformed. TIRFm (Total Internal Reflection microscopy) shows that RNase E in live cells rapidly diffuses over the entire inner membrane forming short-lived foci. Diffusion could be part of a scanning mechanism facilitating substrate recognition and cooperativity. Remarkably, RNase E foci disappear and the rate of RNase E diffusion increases with rifampicin treatment. Control experiments show that the effect of rifampicin is specific to RNase E and that the effect is not a secondary consequence of the shut off of E. coli transcription. We therefore interpret the effect of rifampicin as being due to the depletion of RNA substrates for degradation. We propose a model in which formation of foci and constraints on diffusion arise from the transient clustering of RNase E into cooperative degradation bodies. Recent discoveries that two ribonucleases with major roles in mRNA degradation, RNase E of Escherichia coli and RNase Y of Bacillus subtilis, are localized to the inner cytoplasmic membrane suggest that spatial separation of transcription and mRNA degradation are general features of the bacterial cell. Here we show that RNase E rapidly diffuses over the entire inner membrane forming short-lived foci. Results of molecular dynamics simulations lead us to suggest that RNase E interacts with the lipid membrane by a novel mechanism permitting a high degree of translational freedom. We show that RNA substrate is necessary for the formation of RNase E foci and that formation of foci correlates with constraints on the diffusion of RNase E. We therefore propose that foci are degradation bodies in which several RNase E molecules engage an RNA substrate. The sequestration of the mRNA degradation machinery to the inner cytoplasmic membrane has important consequences for mRNA turnover. This organization likely favors formation of polyribosomes on nascent transcripts before they are exposed to the degradation machinery. Rapid diffusion of RNase E on the inner cytoplasmic membrane could be part of a scanning mechanism that facilitates recognition of cytoplasmic polyribosomes and cooperative degradation of mRNA.