Structural basis of sRNA RsmZ regulation ofPseudomonas aeruginosavirulence

AbstractPseudomonas aeruginosais an opportunistic human pathogen that poses threats to hospitalized immunocompromised patients1. A non-coding small RNA (sRNA) from the repressor of secondary metabolites (Rsm) system, RsmZ, sequesters the global repressor protein RsmA to regulate downstream gene expressions that reprogram virulence repertoires associated with acute and chronicP. aeruginosainfections2,3. Molecular insights into the full-length RsmZ architecture remain elusive, leading to the lack of understanding of RsmZ binding to RsmA and subsequent modulations of gene expressions. Here we use cryo-electron microscopy (cryo-EM) to resolve structures of the full-length RsmZ in complexes with RsmA, in which five stem-loops (SLs) and one single-stranded junction carrying the GGA binding sites in RsmZ form three pairs of clamps, each binding to a RsmA homodimer. Disruptions of the base-pairings in all stems of RsmZ significantly reduced the binding affinity to RsmA by 17-fold, which resulted in enhanced RsmA downregulation of gene expressions and phenotypes associated to both acute and chronic virulence ofP. aeruginosa. Double mutations that rescued these stems of RsmZ restored the binding to RsmA by more than 5-fold, and recovered the corresponding phenotypes. Our results reveal the molecular mechanism of RsmZ regulation ofP. aeruginosavirulence and suggest RsmZ as a potential target for the development of new antimicrobial agents.