The sRNA RyhB Regulates the Synthesis of the Escherichia coli Methionine Sulfoxide Reductase MsrB but Not MsrA
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.