The influence of Escherichia coli Hfq mutations on RNA binding and sRNA•mRNA duplex formation in rpoS riboregulation

The Escherichia coli RNA binding protein Hfq plays an important role in regulating mRNA translation through its interactions with small non-coding RNAs (sRNAs) and specific mRNAs sites. The rpoS mRNA, which codes for a transcription factor, is regulated by several sRNAs. DsrA and RprA enhance translation by pairing to a site on this mRNA, while OxyS represses rpoS mRNA translation. To better understand how Hfq interacts with these sRNAs and rpoS mRNA, the binding of wt Hfq and eleven mutant Hfqs to DsrA, RprA, OxyS and rpoS mRNA was examined. Nine of the mutant Hfq had single-residue mutations located on the proximal, distal, and outer-edge surfaces of the Hfq hexamer, while two Hfq had truncated C-terminal ends. Hfq with outer-edge mutations and truncated C-terminal ends behaved similar to wt Hfq with regard to binding the sRNAs, rpoS mRNA segments, and stimulating DsrA
• rpoS mRNA formation. Proximal surface mutations decreased Hfq binding to the three sRNAs and the rpoS mRNA segment containing the translation initiation region. Distal surface mutations lowered Hfq's affinity for the rpoS mRNA segment containing the (ARN)4 sequence. Strong Hfq binding to both rpoS mRNA segments appears to be needed for maximum enhancement of DsrA
• rpoS mRNA annealing. OxyS bound tightly to Hfq but exhibited weak affinity for rpoS mRNA containing the leader region and 75 nt of coding sequence in the absence or presence of Hfq. This together with other results suggest OxyS represses rpoS mRNA translation by sequestering Hfq rather than binding to rpoS mRNA.

► The binding of mutant Hfqs to sRNAs and segments of rpoS mRNA was examined.
► The ability of mutant Hfqs to enhance sRNA–mRNA binding was examined.
► Hfq with outer edge mutations or truncated C-terminal ends behaved similar to wt Hfq.
► Strong binding of Hfq to both rpoS mRNA sites is needed to enhance sRNA–rpoS mRNA hybridization.
► Results imply OxyS sequesters Hfq rather than binds rpoS mRNA to repress expression.