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. 2021 Jul 22;22(15):7849. doi: 10.3390/ijms22157849

Figure 4.

Figure 4

High-resolution S1-nuclease mapping of the TSS for the SigB-dependent promoters in S. coelicolor A3(2). The 5′-labeled DNA probes were hybridized with 40 μg of RNA and treated with 120 U of S1-nuclease as described in the Materials and Methods. RNA was isolated from liquid cultured S. coelicolor M145 (WT) and its isogenic ΔsigBop and ΔsigHop mutants. The strains grew into the exponential phase (lane 0), then were exposed to osmotic stress conditions with 0.5 M NaCl and 1 M sucrose (suc) and RNA isolated after 30 or 60 min (lanes 30 or 60). E. coli tRNA was used as a control (lane C). Control S1-nuclease mapping was performed with the same RNA samples and a DNA probe for SigH-dependent sigHp2 promoter [27]. The RNA-protected DNA fragments were analyzed on DNA sequencing gels together with G + A (lane A), T + C (lane T) sequencing ladders derived from the end-labelled DNA fragments [46]. The bent arrows indicate positions of the protected fragments.