Cycling of the Sm-like protein Hfq on the DsrA small regulatory RNA.
Small RNAs (sRNAs) regulate bacterial genes involved in environmental adaptation. This RNA regulation requires Hfq, a bacterial Sm-like protein that stabilizes sRNAs and enhances RNA-RNA interactions. To understand the mechanism of target recognition by sRNAs, we investigated the interactions between Hfq, the sRNA DsrA, and its regulatory target rpoS mRNA, which encodes the stress response sigma factor. Nuclease footprinting revealed that Hfq recognized multiple sites in rpoS mRNA without significantly perturbing secondary structure in the 5' leader that inhibits translation initiation. Base-pairing with DsrA, however, made the rpoS ribosome binding site fully accessible, as predicted by genetic data. Hfq bound DsrA four times more tightly than the DsrA.rpoS RNA complex in gel mobility-shift assays. Consequently, Hfq is displaced rapidly from its high-affinity binding site on DsrA by conformational changes in DsrA, when DsrA base-pairs with rpoS mRNA. Hfq accelerated DsrA.rpoS RNA association and stabilized the RNA complex up to twofold. Hybridization of DsrA and rpoS mRNA was optimal when Hfq occupied its primary binding site on free DsrA, but was inhibited when Hfq associated with the DsrA.rpoS RNA complex. We conclude that recognition of rpoS mRNA is stimulated by binding of Hfq to free DsrA sRNA, followed by release of Hfq from the sRNA.mRNA complex.
Lease
Richard A
RA
T. C. Jenkins Department of Biophysics, Johns Hopkins University, 3400 N. Charles St., Baltimore, MD 21218-2865, USA.
Woodson
Sarah A
SA
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics; D020029:Base Pairing; D001483:Base Sequence; D001665:Binding Sites; D018983:DNA Footprinting; D019281:Dimerization; D035001:Host Factor 1 Protein / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D011485:Protein Binding; D011489:Protein Denaturation; D012329:RNA, Bacterial / Q000235:genetics / Q000378:metabolism*; D058727:RNA, Small Untranslated; D022661:RNA, Untranslated / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism; D012808:Sigma Factor / Q000737:chemistry / Q000235:genetics; D013816:Thermodynamics
D001426:Bacterial Proteins; C405431:DsrA RNA, E coli; D035001:Host Factor 1 Protein; D012329:RNA, Bacterial; D058727:RNA, Small Untranslated; D022661:RNA, Untranslated; D012808:Sigma Factor; C069567:sigma factor KatF protein, Bacteria