## 3 The Complex Network of (p)ppGpp Signaling
### Transcriptional Regulation  
One of the earliest discovered effects in the stringent response was selective induction and suppression of bacterial transcriptional promoters [19; 45; 54; 55; 56; 57], which was further confirmed and extended by subsequent transcriptome analysis [58; 59; 60; 61; 62]. Multiple mechanisms have been detected for (p)ppGpp-mediated inhibition of transcription, with direct binding to RNA polymerase (RNAP) being the most straightforward among them, as shown for \(E\). _coli_[63; 64]. Here, instead of binding at the catalytic center or the nucleotide-binding site, (p)ppGpp binds at two regions, one at the interface between the RNA polymerase (RNAP) \(\beta^{\prime}\) and \(\omega\) subunit, and the other between RNAP and the transcription initiation factor, DnaK suppressor A (DksA) [63; 64; 65; 66; 67; 68]. Naturally, the relative binding of (p)ppGpp to the two sites depends on DksA concentration in the cell, such that they function coordinately in different nutritional or environmental conditions [68; 69; 70]. Based on several lines of in vivo and in vitro results, these authors concluded that Site 1 would be preferred when ppGpp concentrations are low, i.e., in nutrient-rich medium, and Site 2 would be preferred when ppGpp concentrations are high enough to fill both sites. This is a noteworthy example of a functional allosteric switch operated by the same ligand binding at two different locations of the same enzyme [71; 72]. In the other mechanism, (p)ppGpp inhibits the binding of the housekeeping sigma factor (\(\sigma\)70) to the RNAP [73], which allows the RNAP to use \(\sigma\)32 and \(\sigma\)38, two alternative sigma factors that are specific for the promoters of genes induced during stress and the stringent response [73]. In Gram-positive bacteria, exemplified by _B. subtilis_, the mechanism is entirely different and more indirect; here, instead of binding to RNAP, (p)ppGpp directly binds to the active site of GMK (guanylate kinase), and the resultant inhibition of GMK reduces the GTP pool [46; 74; 77]. This lowers the transcription from ribosomal RNA promoters, as they initiate with GTP; however, transcription from amino acid biosynthesis operons is induced, mainly because CodY, the repressor of these operons, requires GTP to repress their promoters. Besides _B. subtilis_, this mechanism was evidenced in several other bacteria, including _Thermus thermophilus_, a Gram-negative, thermophilic bacterium [75]. A survey of GMKs from phylogenetically diverse bacteria indeed revealed that the mechanism is conserved in the Actinobacteria, Firmicutes (e.g., _B. subtilis_), and Deinococcus-Thermus, whereas, in the Proteobacteria (e.g., _E. coli_), (p)ppGpp directly regulates RNA polymerase (RNAP) [47]. The authors proposed that GMK is an ancestral (p)ppGpp target, whereas RNAP evolved as a direct target in Proteobacteria only recently [47]. As the direct interaction of (p)ppGpp with RNAP occurs via binding to the interface of \(\beta^{\prime}\) and \(\omega\), it is in effect a tripartite interaction involving \(\beta^{\prime}\), \(\omega\), and ppGpp, which complicates the structure-function correlation studies. In an example, a high-resolution co-crystal structure identified a ppGpp-binding pocket on Thermus thermophilus RNAP [76]. In a complementary study [77], genome-wide transcription initiation sites in _E. coli_ cells were mapped by RNA-seq analysis under two conditions: low basal levels of intracellular ppGpp and at higher levels, produced by expression of recombinant ReIA, which allowed studies of the role of ppGpp in the absence any indirect effect of stress. The results suggested that ppGpp indeed directly interacts with RNAP to transcribe different populations of genes in low and high ppGpp environments, commensurate with the use of two different ppGpp binding sites. Surprisingly, mutations of the ppGpp contact resides on the RNAP failed to abrogate the ppGpp effect, as measured in well-defined RNAP-promoter interactions [75].