## 3 The Complex Network of (p)ppGpp Signaling
### The (p)ppGpp Interactome  
Since the recognition of the stringent response, nearly two dozen ppGpp-interacting proteins have been reported in focused studies of specific effects [53], a few examples of which such as RNAP (Section 3.1), several translational factors (Section 3.2), and DNA primase (Dna G) (Section 3.3) were presented in previous sections, but broader studies revealing many other interactors are discussed here. In an unbiased effort to identify ppGpp-binding proteins, two groups used differential-radial-capillary-action-of-ligand-assays (DRaCALAs) that essentially measure the mobility of radiolabeled ppGpp on a nitrocellulose filter treated with lysates of cells overproducing a single candidate protein [115]. DRaCALA led to the identification of a small number of targets, a total of \(\sim\)30 in Staphylococcus and _E. coli_ added together [115]. This was an encouraging proof-of-concept as the identified candidates included several bona fide proteins, such as five GTPases involved in ribosomal biogenesis and nine known ppGpp-binding proteins [115]. However, if one appreciates the vast functional network of the ppGpp effect, one would perhaps expect a larger number of ppGpp-binding proteins. In fact, as pointed out [116], DRaCALA had missed several proteins that were known to bind ppGpp in vitro, which was conjectured to result from the fact that being a non-equilibrium assay, DRaCALA missed targets with fast off-rates, i.e., some filter-immobilized proteins may have lost the radioactive ppGpp faster than they could bind. Moreover, the need for purified recombinant proteins, which is labor-intensive, limited the number of proteins screened. Recently, multiple additional ppGpp-binders were identified in _E. coli_ by photoaffinity capture [117]. In this method, trifunctional photoaffinity ppGpp compounds are first synthesized, in which ppGpp is coupled with biotin and phenyl azide. The compounds are incubated with extracts of cells that underwent the stringent response, and cross-linking by UV light leads to formation of the covalent bond that prevents dissociation of the proteins. The captured proteins are separated from the lysate by the use of streptavidin-coated magnetic beads. In the last step, the proteins are digested with trypsin and peptides are characterized by standard LC-MS/MS (Liquid Chromatography-Tandem Mass Spectroscopy) to identify the proteins. As a routine practice in such studies, the proteins are finally identified by using the peptide sequences as a query against the genome-wide proteome.  
In a fundamentally similar approach but a more comprehensive expedition, [PERSON] et al. [116] used a similar photoaffinity capture procedure and cross-linkers of different lengths to identify 56 _E. coli_ proteins that directly bind (p)ppGpp, including the previously known ones and several new ones. Interestingly, it also appeared to have missed a few known ppGpp-interactors, such as DNA primase (Section 3.3), the reasons of which are unclear. Nonetheless, several of the newly identified proteins were independently confirmed previously by computer-generated docking [31]. Here, we have paid attention to a few of the remaining hits for which, to the best of our knowledge, in silico protein-ppGpp docking studies have not been reported. The goal here was to obtain structural confirmation of some of the biochemical properties of these proteins, where known, and therefore, we used the same CB-Dock2 program (Supplementary material) that we had used earlier. A cautionary note here is that conclusions derived from in silico structures alone may not be fully reliable; however, they can serve as guides for future research.