# Effects of DNA Topology on Transcription from rRNA Promoters in _Bacillus subtilis_
## 3 Results
### Changes in DNA Topology Affect the Affinity of RNAP for iNTP In Vitro  
To test directly whether DNA topology affects the activity of rRNA promoters, we performed in vitro transcription experiments. We had speculated that the in vivo decrease in the activity of _rrn_B P1 during stationary phase and in response to novobiocin treatment could be due to altered affinity of RNAP for iGTP at this promoter (induced by changes in supercoiling levels): the GTP level does not change but the open promoter becomes less stable, requiring more iGTP for maximal transcription. To address this hypothesis experimentally, we performed in vitro transcriptions with defined components. We used promoter core variants of _rm_B P1 and _Preg_ cloned in the pRLG770 plasmid [11] (for details see Table 1 in Material and Methods section). The DNA templates were used in two different topological forms--in the negatively supercoiled plasmid form (SC), and in the relaxed form (LIN), using the same DNA construct but linearized with the _Psf_I restriction enzyme (Supplementary Figure S3). We performed multiple round transcriptions in vitro with increasing [GTP] (Figure 3). The GTP concentration required for half-maximal transcription (KGTP) was used as a measure of the affinity of RNAP for iGTP at the promoter. A characteristic of rRNA promoters is their requirement for relatively high levels of iGTP for maximal transcription (due to unstable open complexes), reflected in high values of KGTP in vitro. _Preg_, to the contrary, has a low value of KGTP. Experiments with SC templates confirmed previously published results [46], the KGTP for _rrn_B P1 was 277 \(\pm\) 24 uM, and for _Preg_ 36 \(\pm\) 9 uM. Experiments with the LIN templates then revealed that KGTP values for both promoters increased (_rrn_B P1 = 440 \(\pm\) 25 uM, _Preg_ = 511 \(\pm\) 78 uM). In the case of _rm_B P1 the KGTP increased from SC to LIN \(\sim\)1.5x, and in the case of _Preg_ KGTP\(\sim\)14x. Surprisingly, the KGTP value of LIN _Preg_ was even higher than the value for _rm_B P1 (Figure 3B). Importantly, the experiments showed that the strength (the maximal level of transcription) of the _rm_B P1 promoter dramatically decreased on the LIN template whereas in the case of _Preg_ the maximal level of transcription was comparable for both types of the template (Figure 3A, primary data), confirming the hypothesis that DNA relaxation decreases the activity of _rrn_B P1 more than the activity of _Preg_. As the preceding experiments were done with the core version of the _rrn_B P1 promoter, we also decided to use an extended version of the promoter region to assess whether  
Figure 3: The affinity of RNAP for iNTP in vitro changes on different DNA templates. (**A**) Multiple-round transcriptions as a function of GTP concentration: representative primary data and their graphical comparison for _rrn_B P1 core and _Preg_. The maximum signal was set as 1. (**B**) Graphical comparison of KGTP values for SC and LIN DNA templates. The values are calculated from at least four experiments, the error bars show \(\pm\)SD. (**C**) Low affinity for LIN templates of full-length _rrn_ promoter variants. Representative primary data are shown. the surrounding sequence has significant effects. Therefore, we used a DNA fragment containing both _rrn_B P1 and _rrn_B P2 promoters in their native tandem arrangement. Each of them contained their respective native -60 to -40 regions encompassing the UP elements. UP elements are A/T-rich sequences that enhance promoter activity by binding the C-terminal domains of \(\alpha\)-subunits of RNAP [47; 48; 49]. Although their stimulatory effect on rRNA promoters in _B. subtilis_ is less pronounced than, e.g., in _E. coli_ (-30x), it is still significant [11]. Experiments with these promoter versions yielded virtually the same results as with the core version (Figure 3C). The KGTP for _rrn_B P1 (from the tandem promoter fragment) was 242 \(\pm\) 31 \(\upmu\)M for SC and 361 \(\pm\) 46 \(\upmu\)M for LIN.