# Effects of DNA Topology on Transcription from rRNA Promoters in _Bacillus subtilis_
## 2 Materials and Methods
### Promoter Activity Monitored by Quantitative Primer Extension (qPE)  
Promoter constructs were fused to _lacZ_ and activities were assayed by primer extension of the short-lived _lacZ_ mRNA that allows to observe rapid decreases in promoter activity in time. The experiments were conducted as described in [15]. Typically, 1 mL of cells was pipetted directly into 2 mL phenol/chloroform (1:1) and 0.25 mL lysis buffer (50 mM Tris-HCl pH 8.0, 500 mM LiCl, 50 mM EDTA pH 8.0, 5% SDS). After brief vortexing, the recovery marker (RM) was added. The RM RNA was made from _B. subtilis_ strain LK41 as described in [15]. This was followed by immediate sonication. Water was then added to increase the aqueous volume to 6 mL to prevent precipitation of salts, followed by two  
\begin{table}
\begin{tabular}{c c c c c} \hline \hline
**Name** & **Original code** & **Construct** & **Description** & **Reference** \\ \hline \multicolumn{4}{c}{_E. coli_} \\ \hline
**LK2580** & SigE & SigE with C-ter. His6\(\kappa\); BL21(DE3) & This work \\ \hline
**LK1425** & SigF & SigF with C-ter. His6\(\kappa\); BL21(DE3) & This work \\ \hline
**LK1208** & SigH & SigH with C-ter. His6\(\kappa\); BL21(DE3) & This work \\ \hline
**LK2531** & SigN & His-SUMO-SigN in pBM05; BL21(DE3) & This work \\ \hline
**LK1177** & RLG7558 & P\(\it{vgg}\) & pRLG770 with P\(\it{vgg}\) (\(-\)38/+1) +1G; DH5\(\alpha\) & [11] \\ \hline
**LK1522** & RLG7596 & \(\it{rmB}\) P1 core & pRLG770 with \(\it{rmB}\) P1 (\(-\)39/+1); DH5\(\alpha\) & [11] \\ \hline
**LK28** & RLG6927 & \(\it{rmB}\) P1+P2 & pRLG770 with \(\it{rmB}\) P1+P2 (\(-\)248/+8); DH5\(\alpha\) & [15] \\ \hline
**LK17** & RLG6916 & \(\it{rmO}\) P1+P2 & pRLG770 with \(\it{rmO}\) P1+P2 (\(-\)314/+9); DH5\(\alpha\) & This work \\ \hline
**LK1231** & P\(\it{trxA}\) & pRLG770 with \(\it{PtrxA}\) (\(-\)249/+11); DH5\(\alpha\) & This work \\ \hline
**LK1233** & P\(\it{motA}\) & pRLG770 with \(\it{PmotA}\) (\(-\)249/+11); DH5\(\alpha\) & This work \\ \hline
**LK2594** & P\(\it{spolIllD}\) & pRLG770 with \(\it{spolIllD}\) (\(-\)150/+10); DH5\(\alpha\) & This work \\ \hline
**LK1495** & P\(\it{spollQ}\) & pRLG770 with \(\it{spollQ}\) (\(-\)251/+9); DH5\(\alpha\) & This work \\ \hline
**LK1235** & P\(\it{spoVG}\) & pRLG770 with \(\it{spoVG}\) (\(-\)94/+11); DH5\(\alpha\) & This work \\ \hline
**LK2672** & \(\it{sigN}\) P2+P3 & pRLG770 with \(\it{sigN}\) P2+P3 (\(-\)247/+159); DH5\(\alpha\) & This work \\ \hline
**LK2673** & P\(\it{zpab}\) & pRLG770 with P\(\it{zpab}\) (\(-\)266/+175); DH5\(\alpha\) & This work \\ \hline
**LK2608** & P\(\it{zpbjY}\) & pRLG770 with P\(\it{zpbjY}\) (\(-\)304/+155); DH5\(\alpha\) & This work \\ \hline
**LK2609** & P\(\it{zpdG}\) & pRLG770 with P\(\it{zpdG}\) (\(-\)244/+170); DH5\(\alpha\) & This work \\ \hline \hline \end{tabular}
\end{table}
Table 1: \(\it{Cont}\).  
extractions with phenol/chloroform, two precipitations with ethanol, and suspension of the pellet in 20-50 \(\upmu\)L 10 mM Tris-HCl, pH 8.0.  
Primer extension was performed with M-MLV reverse transcriptase as recommended by the manufacturer (Promega) with 1-10 \(\upmu\)L purified RNA. The \({}^{32}\)P 5'-labeled primer (#2973) hybridized 89 nt downstream from the junction of the promoter fragment used for the creation of the _lacZ_ fusion. Samples were electrophoresed on 7 M urea 5.5% or 9% polyacrylamide gels. The gels were exposed to Fuji Imaging Screens. The screens were scanned with Molecular Imager FX (Bio-Rad, Berkeley, CA, USA) and were visualized and analysed using the Quantity One software (Bio-Rad), and normalized to cell number (OD\({}_{600}\)) and RM.