Duplex formation between the sRNA DsrA and rpoS mRNA is not sufficient for efficient RpoS synthesis at low temperature

Abstract

At low temperatures the Escherichia coli rpoS mRNA, encoding the stationary phase sigma factor RpoS, forms an intramolecular secondary structure (iss) that impedes translation initiation. Under these conditions the small RNA DsrA, which is stabilzed by Hfq, forms a duplex with rpoS mRNA sequences opposite of the ribosome-binding site (rbs). Both the DEAD box helicase CsdA and Hfq have been implicated in DsrA·rpoS duplex formation. Hfq binding to A-rich sequences in the rpoS leader has been suggested to restructure the mRNA, and thereby to accelerate DsrA·rpoS duplex formation, which, in turn, was deemed to free the rpoS rbs and to permit ribosome loading on the mRNA. Several experiments designed to elucidate the role of Hfq in DsrA-mediated translational activation of rpoS mRNA have been conducted in vitro. Here, we assessed RpoS synthesis in vivo to further study the role of Hfq in rpoS regulation. We show that RpoS synthesis was reduced when DsrA was ectopically overexpressed at 24 °C in the absence of Hfq despite of DsrA·rpoS duplex formation. This observation indicated that DsrA·rpoS annealing may not be sufficient for efficient ribosome loading on rpoS mRNA. In addition, a HfqG29A mutant protein was employed, which is deficient in binding to A-rich sequences present in the rpoS leader but proficient in DsrA binding. We show that DsrA·rpoS duplex formation occurs in the presence of the HfqG29A mutant protein at low temperature, whereas synthesis of RpoS was greatly diminished. RNase T1 footprinting studies of DsrA·rpoS duplexes in the absence and presence of Hfq or HfqG29A indicated that Hfq is required to resolve a stem-loop structure in the immediate coding region of rpoS mRNA. These in vivo studies corroborate the importance of the A-rich sequences in the rpoS leader and strongly suggest that Hfq, besides stabilizing DsrA and accelerating DsrA·rpoS duplex formation, is also required to convert the rpoS mRNA into a translationally competent form.

Keywords: DsrA; Hfq; riboregulation; rpoS; translational activation.

Figure 1. Hfq/DsrA requirement at 24 °C and 37 °C. The steady-state levels of RpoS were determined by quantitative western blotting in the E. coli hfq^- strain JW4130 harboring the control plasmid pACYC184 (lanes 1 and 3) and plasmid pAHfq, encoding Hfq (lanes 2 and 4), respectively. The strains were grown to early log phase (OD₆₀₀ of 0.4) at 24 °C (lanes 1 and 2) or 37 °C (lanes 3 and 4). Equal amounts of cellular protein were loaded onto the SDS-polyacrylamide gel. Immunodetection of RpoS, ribosomal protein L14 (loading control), and Hfq as well as the detection of DsrA and 5S rRNA (loading control) by northern blot analysis was performed as described in the Materials and Methods. Only the relevant parts of the immunoblots and the autoradiographs are shown.

Figure 2. DsrA·rpoS duplex formation does not lead to efficient translation of rpoS mRNA in the absence of Hfq. (A) Immunodetection of RpoS, L9 ribosomal protein (loading control), and Hfq protein and detection of DsrA and 5S rRNA in the hfq^- strain JW4130 harboring plasmid pNM12 (control; lane 1), pNM13 (encoding DsrA; lane 2), pACYC184 (control; lane 3), and pAHfq (encoding hfq; lane 4), respectively. The proteins and RNAs were visualized as described in the legend to Fig. 1. Only the relevant sections of the immunoblots and autoradiographs are shown. (B) Lanes 1–4, primer extension analysis of total RNA isolated from strains JW4130(pNM12), JW4130(pNM13), JW4130(pACYC184), and JW4130(pAHfq), respectively. The primer extension (PE) signals for rpoS mRNA isolated from the different strains are shown on top. The RNase III-mediated cleavage signals in rpoS mRNA are marked by arrows (G_-112 and A_-109). U, A, C, G, sequencing ladder.

Figure 3. DsrA·rpoS duplex formation in the presence of Hfq_G29A does not result in efficient translation of rpoS mRNA at low temperature. (A) Immunodetection of RpoS, ribosomal protein L9, and Hfq in strains JW4130(pAHfq) (lane 1), JW4130(pAHfq_G29A) (lane 2), and JW4130(pACYC184) (lane 3). DsrA and 5S rRNA were detected as described in the legend to Fig. 1. Only the relevant parts of the immunoblots and autoradiographs are shown. (B) Lanes 1–3, primer extension analysis of total RNA isolated from strains JW4130(pAHfq), JW4130(pAHfq_G29A), and JW4130(pACYC184), respectively. The primer extension (PE) signals for rpoS mRNA isolated from the different strains are shown on top. The RNase III-mediated cleavage signals in rpoS mRNA are marked by arrows (G_-112 and A_-109). G, G sequencing ladder.

Figure 4. Structural probing of the translation initiation region of rpoS mRNA in complex with DsrA in the absence of Hfq and in the presence of Hfq_wt or Hfq_G29A. First, rpoS mRNA was annealed to DsrA and to the [³²P]-5′-end labeled primer. The annealing mix was cooled to 24 °C and divided into three parts followed by incubation for 10 min in the absence and presence of Hfq_wt and Hfq_G29A, respectively. Then RNase T1 was added for 10 min followed by primer extension with AMV reverse transcriptase at 45 °C. The primer extension products were separated on a 8% polyacrylamide-8M urea gel and visualized by a PhosphorImager (Molecular Dynamics). The sequence of the immediate coding region is shown at the left. The start codon is marked by dots. RNase T1 cleavage 3′ of G residues and stop signals of the reverse transcriptase at A residues are indicated at the right. The numbering is given with regard to the A of the start codon (+1). Lane 1, primer extension without RNase T1 digestion. Lane 2–4, structural probing with RNase T1 in the absence of Hfq, in the presence of Hfq_wt and in the presence of Hfq_G29A, respectively. U, A, C, G, sequencing ladder.

Figure 5. Overexpression of csdA does not rescue rpoS translation in the presence of Hfq_G29A. Immunodetection of RpoS, CsdA, Hfq, and ribosomal protein L9 in strains JW4130(pAHfq;pProEx-Htb) (lane 1), JW4130(pAHfq;pCsdA) (lane 2), JW4130(pAHfq_G29A; pProEx-Htb) (lane 3), and JW4130(pAHfq_G29A;pCsdA) (lane 4), respectively. The proteins were visualized as described in the legend to Fig. 1. Only the relevant sections of the immunoblots are shown.

Figure 6. Working model for translational activation of rpoS mRNA at low temperature. The rbs of rpoS mRNA is masked by an iss (in red). In the presence of Hfq (upper scheme) the sRNA DsrA is bound and stabilized by Hfq.^,, CsdA (blue oval) is required for DsrA·rpoS duplex formation, which leads to opening of the iss and in displacement of Hfq from DsrA. Hfq bound to A-rich segments in the rpoS leader (AAYAA) promotes DsrA·rpoS annealing and restructuring of rpoS mRNA into a translationally competent conformer (green bent arrow), which, in turn, permits efficient rpoS translation. In the absence of Hfq (lower scheme), DsrA·rpoS duplex formation occurs when dsrA is overexpressed. However, in the absence of Hfq, rpoS is poorly translated because the local secondary structure within the immediate coding region is not efficiently resolved. The Shine-Dalgarno (SD) sequence and start codon (AUG) in rpoS mRNA are highlighted, Hfq is shown in green.