## 3 The Complex Network of (p)ppGpp Signaling
### Regulation of Replication by (p)ppGpp  
The ability of (p)ppGpp to suppress bacterial cell division was known since the discovery of the stringent response [19]; however, studies of its molecular mechanism have remained scanty. Bacterial DNA replication, essential for cell division, is inhibited by (p)ppGpp both directly and indirectly, depending on bacterial species [97; 98]. In _E. coli_, (p)ppGpp inhibits replication initiation through mechanisms that are dependent on hemimethylation of DNA at the origin of replication (OriC) and binding of the SeqA protein, a negative modulator of replication initiation [99; 100; 98]. Recent studies indicated an underlying mechanism based on the coupling of transcription and DNA replication, modulated by DNA supercoiling [102]. In it, a transcription fork in the vicinity of oriC induces negative supercoiling and DNA strand separation at oriC, which, in turn, facilitates initiation of replication. As rRNA constitutes over 80% of all transcription in a bacterial chromosome and the rRNA loci are clustered near the oriC [103], a global inhibition of transcription by ppGpp during the stringent response could, in principle, diminish the introduction of negative supercoils near oriC, also making the oriC more difficult to melt [104; 105], leading to the observed inhibition of replication initiation. However, [PERSON] et al. [106] provided a strong argument that regulation by ppGpp does not require the ribosomal operons to be present in cis with oriC; instead, ppGpp alters the expression of gyrase and topoisomerase IV, which is responsible for modifying the supercoiling around the origin.  
The stringent response also inhibits DNA elongation [107; 108]. While the effect on gyrase and topoisomerase may also underlie the inhibition, a large body of evidence has established a direct inhibition of DNA primase (DnaG) by (p)ppGpp [107; 108; 109; 110; 111]. DNA primase is a specialized RNA polymerase that catalyzes the synthesis of short RNA molecules,  
Figure 3: In silico docking of ppGpp onto _B. subtilis_ Obg (PDB 1 LNZ). This presentation was generated by PyMol software [95] of the docked complex created by the CB-Dock2 software [96], based on a cavity search followed by optimal ligand-fit, the details of which have been described (Supplementary material). Much of the protein structure has been removed in the display for better visualization of ppGpp-binding, as labeled. The four areas colored red, and designated as G2, G3, G4, G5, are conserved in guanine nucleotide-binding proteins [31; 94] and are also close to the docked ppGpp molecule. The 5\({}^{\prime}\) and 3\({}^{\prime}\) diphosphate chains of ppGpp are colored orange and blue, respectively. The rest of the polypeptide, regardless of secondary structure, is green.  
-11 nt long, used as primers by DNA polymerase at the replication forks in all stages of replication [112; 113; 114]. The (p)ppGpp binding site of primase overlaps with the NTP binding site, and therefore, (p)ppGpp competes with NTP for binding. However, the primase is inhibited by low concentrations of ppGpp (<1 mM) [68; 31] that may be attained early in the stringent response, paralleling the rapid cessation of DNA replication and cell division, further implicating the primase as a key player in the ppGpp effect. Lastly, (p)ppGpp appears to destabilize the association between primase and the RNA-DNA heteroduplex [108].