RNA binding of Hfq monomers promotes RelA-mediated hexamerization in a limiting Hfq environment.

The RNA chaperone Hfq, acting as a hexamer, is a known mediator of post-transcriptional regulation, expediting basepairing between small RNAs (sRNAs) and their target mRNAs. However, the intricate details associated with Hfq-RNA biogenesis are still unclear. Previously, we reported that the stringent response regulator, RelA, is a functional partner of Hfq that facilitates Hfq-mediated sRNA–mRNA regulation in vivo and induces Hfq hexamerization in vitro. Here we show that RelA-mediated Hfq hexamerization requires an initial binding of RNA, preferably sRNA to Hfq monomers. By interacting with a Shine–Dalgarno-like sequence (GGAG) in the sRNA, RelA stabilizes the initially unstable complex of RNA bound-Hfq monomer, enabling the attachment of more Hfq subunits to form a functional hexamer. Overall, our study showing that RNA binding to Hfq monomers is at the heart of RelA-mediated Hfq hexamerization, challenges the previous concept that only Hfq hexamers can bind RNA. RelA stimulates RyhB small RNA–target mRNA interaction by promoting assembly of Hfq monomers into hexamers. Here the authors show that RelA-mediated Hfq hexamerization requires an initial binding of RNA to Hfq monomers.