Effects of Hfq and RNAse E on the steady-state levels of DsrA, RyhB, SsrS, and SsrA RNAs and on the half-life of RyhB. (A) Hfq affects the half-life of RyhB sRNA. Graphical representation of RyhB RNA decay in an hfq⁺ (•) and an hfq⁻strain (▴). The Northern-blot analysis was carried out as described in Materials and Methods using a labeled riboprobe comprising nucleotides 19–60 of RyhB RNA. (B) The steady-state levels of both DsrA and RyhB sRNA are increased in anrne^ts strain at the nonpermissive temperature. The full-length (F) and the truncated (T) forms of DsrA are indicated by arrows on the left. (C) Equal steady-state levels of SsrS RNA in the hfq⁻ and rne^ts strains (lanes 2,4) when compared with the respective wild-type strains (lanes 1,3). (D) The steady-state levels of SsrA RNA in the hfq⁻ strain and in the hfq⁺ strain are equivalent (lanes 1,2), whereas SsrA precursor RNA accumulates upon inactivation of RNase E (cf. lanes 3,4). (B,C,D, bottom) Detection of 5S rRNA (loading control). The Northern-blot analysis and detection of sRNAs in total RNA was performed as described in Materials and Methods.

Hfq protects ompA mRNA from RNase E cleavage at site D. (A) Primary structure of the 5′UTR of ompA180 mRNA with the 5′-terminal stem–loop structures (hp1 and hp2) and the 5′-initial coding region (structure III, in Rosenbaum et al. 1993). The mRNA contains the 5′UTR and the first 45 nucleotides of the ompA gene. The A/U-rich sequence ss2 is depicted by a bar. The SD sequence, as well as the start codon (Met), are indicated by bars and the positions of the RNase E cleavage sites C and D are depicted by arrows. The ompA117, ompα117Δhp2, and ompA 117Δss2 mRNAs used in the gel mobility-shift assays (see C) are depicted by hatched bars. (B) In vitro cleavage of ompA180 mRNA in the presence or absence of Hfq. [³²P]-5′-end labeled ompA180 mRNA was incubated in RNase E cleavage buffer at 37°C without (lane 1) or in the presence of a degradosome preparation (lanes 2–4). Hfq-hexamer [Hfq(6)] was added in a fourfold (lane 3) and 12-fold (lane 4) molar excess over ompA180 mRNA prior to addition of the RNase E preparation. The RNase E cleavage reaction was carried out for 30 min at 37°C. Cleavage at sites C and D (see A) by the degradosome preparation is indicated at right. (C) Binding of Hfq to ompA117, ompA117Δhp2, and ompA117Δss2 mRNAs. The 5′end-labeled mRNAs were incubated with purified Hfq protein to allow formation of the RNA–protein complex as specified in Materials and Methods, and then resolved on a 4% native polyacrylamide gel. (Lanes 1,3,5) Electrophoretic mobility of ompA117, ompA117Δhp2, and ompA117Δss2 mRNAs, respectively; (lanes 2,4,6) electrophoretic mobility of ompA117, ompA117Δhp2, and ompA117Δss2 mRNAs, respectively, in the presence of a fourfold molar excess of Hfq-hexamer.

Hfq protects DsrA and RyhB sRNAs from RNase E cleavage. (A) The secondary structure of DsrA RNA was determined by Brescia et al. (2003). A ribo-oligonucleotide corresponding to Domain II (DII) inhibited binding of Hfq to DsrA (Brescia et al. 2003). The position of the RNase E cleavage site is indicated by an arrow. SL1, SL2, and SL3 denote the three stem–loop structures. (B) In vitro cleavage of DsrA RNA in the presence and absence of Hfq. [³²P]-5′-end labeled DsrA was incubated in RNase E cleavage buffer at 37°C without (lane 1) or in the presence of a degradosome preparation (lanes 2–5). Hfq-hexamer was added in a fourfold (lane 3), 12-fold (lane 4), and 20-fold (lane 5) molar excess over DsrA prior to addition of the RNase E preparation. The RNase E cleavage reaction was carried out for 3 min at 37°C. (C) Secondary structure of RyhB RNA as revealed by Massé and Gottesman (2002) using bioinformatics. The RNase E cleavage site is indicated by an arrow. (D) In vitro cleavage of RyhB RNA in the presence and absence of Hfq. [³²P]-5′-end labeled RyhB was incubated in RNase E cleavage buffer at 37°C without (lane 2) or in the presence of a degradosome preparation (lanes 3–6). Hfq-hexamer was added in a fourfold (lane 4), 12-fold (lane 5), and 20-fold (lane 6) molar excess over RyhB prior to addition of the RNase E preparation. The RNase E cleavage reaction was carried out for 3 min at 37°C. (Lane 1) RNase T1 ladder.