RNase III autoregulates its own expression.
(A) Visualization of mapped cDNA reads on the S. aureus genome corresponding to rnc mRNA fragments using the Integrated Genome Browser (IGB, Affymetrix). Same legend as described in Figure 2A. The red arrow indicates the start of a cleaved fragment at position +U296. (B) In vitro RNase III cleavage assays on unlabeled full-length rnc mRNA (FL-rnc 843 nt, lanes 1–7) or the rnc mRNA lacking its 5′ untranslated region (Δ5′UTR-rnc 752 nt, lanes 8–12). The RNA fragments were separated using an agarose gel under denaturing conditions and visualized after ethidium bromide staining. Unlabeled rnc mRNA (200 nM) was incubated with purified wild type RNase III (WT) (lanes 2, 3, 9, 10) or the mutants E135A (lanes 4, 5, 11) or D63A (lanes 6, 7, 12). Lanes 1, 8: incubation controls of rnc mRNAs in the absence of enzyme. Cleavage assays were performed with WT-RNase III at 0.33 µM in a buffer containing Mg2+ (lanes 2, 9) or Ca2+ (lanes 3, 10), with the mutant E135A at 0.33 µM (lanes 4, 11) and 0.66 µM (lane 5), and with the mutant D63A at 0.33 µM (lane 6, 12) and 0.66 µM (lane 7). Lane M: Riboruler low range RNA marker (Fermentas). (C) RNase III cleavage sites were mapped on in vitro transcribed rnc mRNA using primer extension with the 5′ end-labeled oligonucleotide 69 (Table S8). Reactions were performed in the presence of increasing concentrations of wild type RNase III (0.33 and 0.66 µM) in a buffer containing either Mg2+ or Ca2+. Lanes U, C, G, A: DNA sequencing reactions but the labels correspond to the RNA sequence. The black arrow denotes the RNase III cleavage at U+296. (D) Secondary structure of the RNase III-binding site located in the coding region of rnc mRNA. A black triangle indicates the in vitro cleavage site at position U+296 while the red arrow represents the primer extension stop.