A Nitric Oxide Regulated Small RNA Controls Expression of Genes Involved in Redox Homeostasis in Bacillus subtilis
Figure 9
RNase III cleaves the ppnKB/RoxS duplex in vitro.
(A) Summary of structure probing experiments showing proposed secondary structure around the translation initiation site of the ppnKB mRNA. The Shine and Dalgarno (SD) sequence is shown in red. The legend for the different cleavages is given under the schematic. (B) A proposed duplex formed between ppnKB (black) and full-length RoxS (green). The ppnKB Shine and Dalgarno sequence is shown in red. The legend for the different cleavages is the same as panel A. The CRR regions 1–3 of RoxS are indicated. The sites of protection from RNase T1 cleavages at −12/13, −3/4, and +2/3 upon duplex formation are encircled (S7 Fig.). RoxS induced RT stops are marked with an asterisk. (C) Proposed duplex formed between ppnKB (black) and truncated RoxS (green). Legend as in panel B. (D) Autoradiograph of in vitro RNase III cleavage assays showing sites of RNase III cleavage (double-headed red arrows) in ppnKB bound to full-length (WT) or various mutant or truncated forms of RoxS. The 5′ ends of primer extension products resulting from RNase III cleavage of ppnKB alone are identified to the right of the gel relative to the first nt of the AUG start codon (double-headed red arrows with circle). Note that cleavage sites are by convention identified by the nt immediately upstream of the corresponding primer extension product. The RT stops at positions −9/10 provoked by RoxS binding to ppnKB and the new RNase III cleavages at positions −12/13, seen upon duplex formation, are indicated to the left of the gel (double-headed red arrows). The Shine and Dalgarno sequence is indicated by SD. (++) indicates addition of twice the quantity of RoxS (80 nM vs. 40 nM) as in lanes marked with (+).