# Effects of DNA Topology on Transcription from rRNA Promoters in _Bacillus subtilis_
## 1 Introduction  
Bacterial cells need to adapt to environmental changes. In nutrient-rich environments, cells grow and divide rapidly, and this requires a large number of ribosomes to satisfy the need for new proteins. In nutritionally poor environments, the synthesis of new ribosomes stops. As the production of new ribosomes is energetically costly for the cell, it must be tightly regulated. The number of ribosomes in the cell is regulated mainly on the level of transcription initiation of ribosomal RNA (rRNA) [1]. Transcription initiation can be divided into several steps. First, when the RNA polymerase (RNAP) holoenzyme (the core RNAP subunits [\(\alpha 2\beta\beta\cdot\omega\)] in complex with a \(\sigma\) factor) binds to specific DNA sequences, promoters, it forms the closed complex where DNA is still in the double-helical form [2]. The specificity of RNAP for promoter sequences is provided by the \(e\) factor [3; 4; 5; 6]. Subsequently, this complex isomerizes and forms the open complex where the two DNA strands are unwound, and the transcription bubble is formed. At this stage, initiating nucleoside triphosphates NTPs (iNTPs) can enter the active site and transcription can begin. RNAP then leaves the promoter and enters the elongation phase of transcription [7]. In bacteria, the concentrations of iNTPs act as key regulators of transcription and directly affect RNAP at some promoters. These promoters form relatively unstable open complexes where the time window available to iNTPs to penetrate into the active site and initiate transcription is relatively short. The higher the concentration of the respective iNTP, the higher the chance that it penetrates into the active site while the transcription bubble isstill open. Hence, increases in intracellular concentrations of iNTPs stimulate transcription whereas low levels of iNTPs result in inefficient transcription initiation [8; 9; 10]. rRNA promoters are prime examples of where transcription initiation is regulated by the concentration of the iNTP. In _Bacillus subtilis_, a model soil-dwelling, spore-forming Gram-positive bacterium, the iNTP of the tandem rRNA promoters of all 10 rRNA operons is exclusively GTP [11]. The GTP level in _B. subtilis_ is affected by (p)ppGpp, an alarmone that is produced at times of stress, such as amino acid starvation or heat shock. (p)ppGpp inhibits GuaB, the first enzyme in the de novo GTP biosynthesis pathway, which results in decreased GTP levels and increased ATP levels as more of the last common intermediate for the synthesis of both GTP and ATP, inosine monophosphate (IMP), is now available for ATP synthesis only [12; 13]. By affecting the GTP level (p)ppGpp indirectly affects the activity of rRNA promoters in _B. subtilis_[14; 15; 16]. This might be similar in other Gram-positive microorganisms, such as _Staphylococcus aureus_, where the GTP concentration ([GTP]) affects rRNA promoter activity under stringent conditions [17]. Another important factor for transcription initiation in bacteria is the topological state of DNA, i.e., the levels of supercoiling. DNA in the cells is usually underwound and this results in negative supercoiling [18]. Negative supercoiling then helps RNAP to melt DNA in promoter regions. In general, bacterial cells display more pronounced negative supercoiling in exponential than in stationary phase of growth and initiation from a number of promoters is sensitive to this parameter [19; 20; 21; 22; 23]. Here, we investigated how the activity of rRNA promoters in _B. subtilis_ changes when the cells transition from exponential to stationary phase. These promoters depend on the primary \(\sigma\) factor, \(\sigma^{\Lambda}\). We show that their activity decreases during the transition and this correlates with a decrease in the GTP concentration. Nevertheless, there is a point in the process where the level of GTP does not decrease any further but the activity of rRNA promoters does. We show that besides [GTP], _B.