# Novel Aspects of Polynucleotide Phosphorylase Function in _Streptomyces_
## 5 (p)ppGpp as a PNPase Effector  
Highly phosphorylated guanine nucleotides, (p)ppGpp, guanosine pentaphosphate, and guanosine tetraphosphate, are alarmones that play a number of roles in the regulation of bacterial metabolism (reviewed in [50; 51]). (p)ppGpp is synthesized by the product of the _relA_ gene in _E. coli_[52], and that gene is found in _S. coelicolor_[53; 54], _S. antibiotic_[55], and other streptomycetes [56]. In _Streptomyces_, ppGpp plays an important role in the regulation of antibiotic synthesis [53; 54; 55; 56]. PNPase from _S. antibiotic_ was shown to synthesize pppGpp in vitro [57; 58]. While this activity may be an in vitro artifact, as no other PNPases are known to possess it, the observation suggested a possible relationship between (p)ppGpp and RNA decay in _Streptomyces_. To begin to examine this relationship, the effects of (p)ppGpp on polymerization and phosphorylosis by _S. coelicolor_, _S. antibiotic_, and _E. coli_ PNPases were measured in the absence and presence of (p)ppGpp [59]. As shown in Figure 6, both guanosine penta- and tetraphosphates inhibited the activity of _S. coelicolor_ PNPase, in both phosphorylosis and polymerization, though ppGpp was a more potent inhibitor than ppGpp. Essentially identical results were obtained for _S. antibiotic_s PNPase (not shown). By contrast, neither ppGpp nor ppGpp were effective inhibitors of the activity of _E. coli_ PNPase. Indeed, at concentrations up to 1 mM, ppGpp actually stimulated the polymerizing activity of the _E. coli_ enzyme. Figure 5: Model for the effects of NDPs on the activity _S. coelicolor_ PNPase. The model posits that _S. coelicolor_ PNPase (PacMan symbol) is able to phosphorolyze 5650 and other structured substrates to a limited extent in the absence of NDPs, as indicated by the dashed X. In the presence of NDPs, PNPase synthesizes unstructured 3′-tails in vivo, and these tails then provide an anchor for the enzyme, thus facilitating the digestion of structured substrates. Copyright © American Society for Microbiology (_[PERSON]_ 195, 2013, 5151–5159, DOI:10.1128/JB.00936-13) [49]. In the same study, the effects of (p)ppGpp on the stability of bulk mRNA in _S. coelicolor_ were examined [59]. It was initially observed that the half-life of bulk mRNA increased by 1.8-fold in stationary phase cultures as compared with exponential phase. That this increase might be related to the effects of (p)ppGpp was suggested by studies with an _S. coelicolor relA_ mutant, and a strain containing an inducible _relA_ gene. While the half-life of bulk mRNA was longer in the _relA_ mutant than in the parental strain (e.g., 8.9 min vs 3.2 min in exponential phase), the half-life decreased slightly in the _relA_ mutant in stationary phase (to 7.2 min). In the strain containing an inducible _relA_ gene, producing increased levels of (p)ppGpp, induction occasioned a ca. two-fold increase in the half-life of bulk mRNA, from 6.6 to 11.8 min. Taken together, these observations suggest that (p)ppGpp may stabilize mRNAs in stationary phase _S. coelicolor_ cells, as compared with cells growing exponentially. Why and how might this stabilization occur? It is well established that although levels of RNA and protein synthesis decrease dramatically as _Streptomyces_ cultures move from the exponential to the stationary phase of growth, a basal level of synthesis is maintained throughout stationary phase [60; 61]. This basal level of macromolecular synthesis is presumably required to produce enzymes and other proteins involved in the synthesis of the secondary metabolites these organisms produce in stationary phase. Stabilization of the transcripts for these proteins would represent one strategy the organisms could employ to ensure the persistence of macromolecular synthesis to support secondary metabolite production. It is known that (p)ppGpp is present in significant amounts, even in stationary phase streptomycte cultures [62; 63].