# The _Ins_ Gene of _Escherichia coli_ Is Transcriptionally Down-Regulated by (p)ppGpp
## 4 Discussion
Although not being conclusive, given the limited number of genes tested, this study permitted the identification of some DNA sequence signatures characteristic of the three classes of promoters: inhibited, activated, and unaffected. Thus, the sequence of _lns_ promoter was superimposed to the histograms of base distributions for promoters inhibited by (p)ppGpp as determined by [PERSON] et al. [11]. This comparison, illustrated as bar graphs in Figure 6, includes the \(-10\) promoter element (the TATA box) and the transcriptional start site (TSS) region encompassing the discriminator (from position \(-1\) to position \(-6\) and the initial transcribed sequence. Concerning the -10 consensus sequence, although three bases, the thymines at positions \(-12\) and \(-7\) and the adenine at position \(-11\), are poorly informative due to the high sequence conservation of TATA box in all promoters, _lns_ shows an adenine at position \(-8\) that was found to interact with RNAP [59] and is strongly favored in promoters negatively affected by (p)ppGpp (Figure 6B). Furthermore, the cytosine at position \(-13\) (extended \(-10\) region) and the spacer length of 17 bp are compatible with promoters repressed by (p)ppGpp (Figure 6A). The discriminator region with its high G+C content is a potential disadvantage to strand-separation at transcription initiation and has been long related with (p)ppGpp down-regulation [60]. Relatively to _lns_ sequence, three cytosines occupy positions \(-4\), \(-3\) and \(-1\), respectively, thus contributing to constitute a robust discriminator (Figure 6C). Notably, the cytosine at position \(-1\) was found at very high frequency (\(\sim\)85%) in inhibited promoters since its presence is required to stabilize the incoming rNTP. Conservation of C-1 is crucial given that it reduces the abortive products formation and accelerates the RNAP escape from those promoters as the ones negatively controlled by (p)ppGpp that show short-lived open complexes [61; 62]. According to these findings, band shift assays clearly indicate that the stability and/or formation of _lns_ promoter-RNAP complexes is impaired by the presence of the modified nucleotide (Figure 5), given an explanation for the (p)ppGpp-dependent reduction in the in vitro transcriptional activity of _lns_ promoter (Figure 4). Furthermore, the _lns_ sequence analysis reveals that the initial rNTPs incorporated into _lns_ mRNA (Figure 6C, positions A+2, C+3 and A+6) well match with transcripts repressed by this alarmone. Regarding the \(-35\) consensus hexamer, no significant sequence difference has been observed between activated and inhibited promoters suggesting that this element does not actively participate to (p)ppGpp regulation [11]. Summarizing, the _lns_ promoter exhibits the primary sequence determinants accounting for a direct regulation by (p)ppGpp as validated through in vitro experiments (Figures 4 and 5). In addition to (p)ppGpp, a second effector, playing a crucial role during the stringent response, is the transcription factor DksA. This small protein (17 kDa) binds RNAP through the secondary channel [63] and largely contributes to create, at protein-protein contact region, the (p)ppGpp binding site 2 [10]. Differently, site 1 is located on RNAP at the interface between \(\beta\)' and \(\omega\) subunits and does not need additional proteins to be formed [9]. DksA was found to function in synergy with (p)ppGpp strengthening its effects, either positive or negative, on transcription. Thus, DksA and (p)ppGpp together work better than either alone and up- or down-regulation depends on the intrinsic kinetic properties of the distinctive promoter controlled by (p)ppGpp [7; 64; 65; 66; 67]. In this context, there is no evidence that the preparations of RNAP, both the commercial and purified one in our laboratory used in in vitro assays (Figures 4 and 5), contain appreciable amounts of DksA, and as a consequence these enzymes lack the (p)ppGpp binding site 2.