Table 1.
List of oligonucleotides used in this study.
Table 2.
List of bacterial strains and plasmids used in this study.
Table 3.
Expression of msrA-lacZ and msrB-lacZ fusions in wild type, ryhB deletion or fur deletion strains grown to exponential phase (O.D.600≈0.4) in LB rich medium either in the presence or in the absence of iron chelator (2,2′dip, 250 µM).
Figure 1.
RyhB-dependent down-regulation of msrB mRNAs and proteins.
(A) Cultures containing a wild type strain of E. coli were grown to an O.D.600 value of 0.5 then 250 μM 2,2′dip was added. After 30 min of incubation with 2,2′dip, total RNA and proteins were extracted in parallel, giving total RNA used for the Northern blots (top panel) and soluble protein fraction used for Western blot (bottom panel). The same membrane was probed successively for msrB mRNA, RyhB, and 23S RNA (loading control) (top panel). MsrB proteins were probed with anti-HA antibodies (bottom panel). The radioactive probes used are described in Materials and Methods, and Table 1. (B). Quantification of msrB mRNA, RyhB sRNA and MsrB protein levels (arbitrary units) from experiment described in (A). Band intensity was normalized to that of an internal control (23S for both msrB and RyhB RNA bands; a non-specific protein recognized by anti-HA antibodies for MsrB-HA protein band). (C). Overview of the experiment described in D. Total RNA was extracted at the indicated times (min). (D). Wild type E. coli cells were grown in LB (lanes 1,2,5), LB + 2,2′dip (250 μM) (lanes 3,4). Iron (100 μM) was added after 15 min of growth in LB (lane 8) and after 5 or 15 min of pre-incubation with 2,2′dip (lanes 6–7). Samples were removed at indicated time points, and total RNA was extracted as described in Materials and Methods. Strain SMG505 (ΔmsrB) was used as a control (lane 1). For determination of RyhB and msrB RNA amounts, 10 μg of total RNA samples were loaded onto a denaturating agarose gel. After migration, a Northern blot hybridization was performed with a specific oligoprobe for RyhB and msrB respectively. Quantification of msrB and RyhB transcript levels (arbitrary units) are shown below Northern blots panels.
Figure 2.
Effects of ryhB, fur, rne701 and hfq mutations on msrB transcript stability.
msrB transcript stability in wild type (A), ryhB mutant (B), fur mutant (C), hfq mutant (D) and rne701 mutant (E) strains grown at 37°C to an O.D.600 of 0.4, was assayed by Northern blot analysis. After 10 min of incubation with 2,2′dip, rifampicin was added. Samples were removed at the indicated time points after rifampicin addition and total RNA was extracted as described in Materials and Methods. For determination of msrB mRNA amount, 10 μg of total RNA samples were loaded onto a denaturating agarose gel. After migration, a Northern blot hybridization was performed with a specific oligoprobe for msrB and 5S as an internal control. Band intensity of msrB transcript was normalized to that of 5S RNA. Half-life (seconds) of msrB transcript and the ratio of msrB mRNA half-life ±2,2′dip are indicated for the different strains. Standard errors (SE) are shown.
Figure 3.
RyhB binds msrB mRNA at two sites.
Panel (A) shows the predicted interactions between RyhB and the msrB sense strand referred as Site I and Site II (in yellow). The predicted ribosome-binding site for msrB is underlined. The start codon for msrB is shown underlined and in italics. Mutations in Site I and Site II are shown in red and blue respectively. (B) cDNA extension experiments with wild type msrB1–124 as a template for the reverse transcriptase. Lane 1: extension with no other component added; lane 2: extension with RyhB alone. C, U, A, and G are sequencing lanes obtained using the same radiolabeled primer as in the reverse extension analysis. Reverse transcriptase stops are indicated at positions +11, +33 and +34. Nucleotides involved in Sites I and II are indicated as thin vertical lines. The transcription start of msrB is referred to as the position + 1. The numbers on the left indicate sequence positions with respect to the transcription start.
Figure 4.
Changes in msrB RNA accessibility to enzymatic and chemical probes upon RyhB binding.
(A) RNases and lead (II) footprinting: 5′ end-labelled msrB1–124 transcript was subjected to partial digestion with RNase T2 (lanes 3–4), RNase V1 (lanes 5–6) or lead (II) (lanes 7–8) in the presence (+) or in the absence (-) of RyhB sRNA. Lanes 1 and 2 are control lanes of RT extension on msrB alone (lane 1) or on msrB with RyhB (lane 2). The resulting fragments were then analyzed onto a denaturing sequencing gel. The numbers indicate sequence positions with respect to the transcription start site. Lanes OH− and T1 correspond, respectively, to an alkaline hydrolysis ladder, and an RNase T1 digestion ladder obtained in denaturing conditions. The position of G residues that resulted from RNase T1 hydrolysis is given. Circles, arrowheads, and rectangles indicate, respectively, phosphodiester bonds cleavages by RNase T2, RNase V1, and lead (II). Products resulting from a strong (red) or a weak (orange) enhancement of the cleavages in presence of RyhB are indicated. Reduced levels of cleavages in presence of RyhB are indicated by dark green (strong) and light green (weak) symbols. RyhB-binding sites (Sites I and II) are shown as thin vertical lines. (B). Summary of the RNases/lead (II) footprints of msrB1–124 mRNA in the presence of RyhB based on the results obtained in (A). The translation start codon of msrB is shown in bold and the Shine Dalgarno sequence is underlined. RyhB Stem Loop 2 (SL2) pairing at Site I and Site II is shown. The same rules as in panel A are utilized for representation of changes in phosphodiester bonds cleavages in presence of RyhB.
Figure 5.
Mutagenesis analysis of the RyhB/msrB interaction in vitro.
(A–B) Autoradiograms of primer extension analysis of msrB mRNA are shown (for details, see Materials and Methods, and Results). (A) with wild type (lanes 1–2), mut2a (lanes 3,4) and mut2b (lanes 5,6) msrB1–124 transcripts as a template for the reverse transcriptase. Lanes 1,3 and 5: extension with no other component added; lanes 2,4 and 6: extension with RyhB. C, U, A, and G are sequencing lanes obtained using the same radiolabeled primer as in the reverse extension analysis. (B) cDNA extension experiments with wild type (lanes 1–2) and mut1 (lane 3) msrB1–124 transcripts as templates for the reverse transcriptase. Lane 1: extension with no other component added; lanes 2–3: extension with RyhB. For (A) and (B), reverse transcriptase stops are indicated at positions +11, +33 and +34. Thin vertical lines indicate nucleotides involved in Site I and Site II. The transcription start of msrB is referred to as the position + 1. The numbers to the left indicate sequence positions with respect to the transcription start site.
Figure 6.
RyhB binding at Site I and Site II blocks ribosome binding to the msrB translation initiation region.
(A) An autoradiogram of a toeprint analysis is shown (for details, see Materials and Methods, and Results). msrB1–450 wild type and variants (mut1 and mut2b) were used as a template (5 nM) in the cDNA extension experiment. Lanes (1, 5, 9): extension with no other component added; lanes (2, 6, 10): extension with RyhB alone (2.5 μM); in lanes (3, 7, 11): extension with 30S subunit (+initiator tRNAfmet) alone (500 nM) ; lanes (4, 8, 12): cDNA extension with 30S subunits (500 nM) along with RyhB (2.5 μM). Thin vertical lines indicate nucleotides involved in Site I and Site II. The transcription start of msrB is referred to as the position + 1. The 30S subunit-induced reverse-transcriptase (RT) toeprint is indicated at positions +50 to +52. Other indicated positions are numbered accordingly. (B) Quantification of band intensity was performed by using Image J software and expressed in terms of fold change in toeprint intensity. Fold change represents decrease in toeprint intensity obtained when comparing the two conditions, with 30S ribosomal subunits alone and with 30S ribosomal subunits along with RyhB. Standard error of two independent experiments is shown.
Figure 7.
Effect of mutations in RyhB-binding Sites I and II on msrB mRNA stability.
Northern blot analysis of wild type msrB (A), msrB mut1 (B), msrB mut2a (C), msrB mut1,2a (D), msrB mut2b (E), and msrB mut1,2b (F). Strains were grown at 37°C to an O.D.600 of 0.4. After 10 min of incubation with 2,2′dip, rifampicin was added. Samples were removed at the times indicated after rifampicin addition and total RNA was extracted as described in Materials and Methods. Half-life of msrB mRNA was calculated with or without iron chelator. For determination of msrB mRNA amount, 10 μg of total RNA samples was loaded on a denaturating 1.2% agarose gel. After migration, a Northern blot hybridization was performed with a specific oligoprobe for msrB and with 5S as a loading control. Half-life (seconds) of msrB mRNA (wild type and mutants) and the ratio of msrB mRNA half-life ±2,2′dip, are indicated. Band intensity of msrB transcript was normalized to that of 5S RNA.