# Effects of DNA Topology on Transcription from rRNA Promoters in _Bacillus subtilis_
## 4 Discussion
### rRNA Promoters and Preg  
In our experiments, the drop in rRNA promoter activity during transition to stationary phase was pronounced and concurrent with the onset of stationary phase. A decrease in the activity of _B. subtilis_ rRNA promoters in stationary phase was also observed in [62]. However, they used promoter constructs fused with GFP and monitored promoter activity by measuring the intensity of fluorescent signal. GFP is relatively stable, so the decreases they reported were less pronounced than those observed in our experiments. Here, we propose an updated model of regulation of _B. subtilis_ rRNA promoters, revealing supercoiling as a factor involved in their control. The more negatively supercoiled DNA in exponential phase contributes to the high activity of _B. subtilis_ rRNA promoters. As this negative supercoiling becomes more relaxed when the cell transitions into stationary phase, this likely contributes to the decrease in the activity of RNAP at rRNA promoters. This is in part due to the decreased affinity of RNAP at rRNA promoters for the initiating GTP but also to a so far unknown step during transcription initiation (e.g., isomerization, promoter escape). The activity of rRNA promoters in stationary phase is also likely affected by the decreased RNAP concentration. The decrease in the available RNAP pool is further exacerbated by the association of the RNAP:\(\sigma^{\text{A}}\) holoenzyme with 6S-1 RNA that sequesters  
Figure 6: Transcription in vitro with alternative \(\sigma\) factors on different DNA templates. Representative primary data are shown (radioactively labelled transcripts resolved by polyacrylamide electrophoresis). SC stands for supercoiled promoter DNA, LIN for linear DNA. Letters above the gels indicate the sigma factor used—A for \(\sigma^{\text{A}}\), B for \(\sigma^{\text{B}}\) etc. Each sigma factor is depicted with different color (\(\sigma^{\text{A}}\), black; \(\sigma^{\text{B}}\), dark blue; \(\sigma^{\text{D}}\), green; \(\sigma^{\text{E}}\), yellow; \(\sigma^{\text{F}}\), brown; \(\sigma^{\text{H}}\), red and \(\sigma^{\text{N}}\), purple). For each promoter three independent experiments were performed. Transcription from SC was set as 1 for each promoter. Quantitation of results is shown in graphs below the respective primary data. The graphs show averages \(\pm\)SD. The reactions with \(\sigma^{\text{A}}\) on all promoter fragments were used to show that the observed transcription was due to the addition of the specific \(\sigma\) factors and not due to (theoretical) contamination of the core with \(\sigma^{\text{A}}\). (**A**) Transcription in vitro from selected \(\sigma^{\text{B}}\), \(\sigma^{\text{D}}\), \(\sigma^{\text{E}}\), \(\sigma^{\text{F}}\) and \(\sigma^{\text{H}}\) -dependent promoters. (**B**) Transcription in vitro from \(\sigma^{\text{N}}\)-dependent promoters. (**C**) Transcription in vitro using a longer construct, _sigN_ P2+P3. P2 is \(\sigma^{\text{A}}\)-dependent, P3 is \(\sigma^{\text{N}}\)-dependent. it in an inactive form in stationary phase [63]. The combined effect results in the shut-off of rRNA synthesis. Previously, for _E. coli_ rRNA promoters, the decreased stability of the open complex was identified as the main kinetic intermediate affected by supercoiling [64]. We also note that in _S. aureus_ in post-exponential growth phase the downregulation of rRNA is independent of ppGpp or NTP pools [17], and it is possible that DNA topology might be a factor contributing to this downregulation. In _B. subtilis_, correlations between the supercoiling level and rRNA activity could be found also in the forespore. Within the developing spore, DNA becomes more negatively supercoiled then in stationary phase [40] and this correlates with an increase in rRNA activity in the forespore [62]. Interestingly, during novobiocin treatment the GTP level increases in _B. subtilis_ and the changes in DNA topology override its stimulatory effect so that the net result is a decrease in the activity of _rm_B P1.