## 5 Unanswered Questions and Future Directions
### Replacement of Stringent Response by Stringent-like Response  
The general disappearance of the classic (p)ppGpp-based stringent response in the evolution of life from unicellular to multicellular organisms is an enigma, but we can try to formulate a scenario based on the genetics and the survival needs of the extant organisms, which is schematically depicted here (Figure 8). It goes without saying that evolutionary models are often fragmented with missing pieces, and the stringent response is no exception. However, we hope that the discussions here will invite refinements, alternative models, and experimental verifications, where possible. The paucity of (p)ppGpp in the metazoa also coincides with the apparent lack of recognizable ReLA homologs, especially in the higher metazoa, such as mammals [135; 136; 151]. However, as stated in the Introduction, there is evidence of barely detectable amounts of (p)ppGpp in some metazoa, but mostly in the evolutionarily earlier species, which makes it tempting to speculate that the early metazoa were at the transition from the (p)ppGpp- to NADPH-based response. Studies of more organisms in the boundary between prokaryotes and primitive eukaryotes may unearth various stages of evolution of the stringent response. The structural similarity between ppGpp and NADPH, particularly in the purine nucleotide parts, has led to the suggestion of their functional equivalence [135]. Both alarmones were/are produced by enzymatic conversion of existing compounds, namely GTP/GDP and NADP/NADPH, which are indispensable for cellular housekeeping functions. In contrast, our knowledge of the role of their interactome is woefully incomplete, as most polypeptides in this group are of unknown function. It is unlikely that all of them simultaneously acquired the ability to bind an alarmome; a more plausible scenario is gradual evolution of small subsets of proteins, initially harboring primordial binding domains that were perfected for optimal activity over time (Figure 8). The previously mentioned evidence that small basal levels of (p)ppGpp have important functions [30; 152]  
Figure 8: Tentative theory of evolution of the stringent response in prokaryotes (**A**) and the very different response in metazoa, dubbed ‘stringent-like response’ (Section 4) (**B**). This is only a schematic of the biochemical pathways that have been described in this review, integrated with possible steps of evolutionary expansion of the interactome (hereby named ‘stringentome’). In Panel A, we propose four alternative arrangements (1–4) of (p)ppGpp– and GTP-binding pockets in a proximal signaling protein of the interactome, as depicted schematically. and that the affinity of (p)ppGpp-binding proteins can vary over three orders of magnitude, i.e., from micro- to milli-molar concentrations of (p)ppGpp [31; 32; 38; 93; 116], suggest that the binding pockets that occur in modern (extant) cells have been stabilized for their respective K\({}_{\text{m}}\) at this stage of evolution. Evolutionary changes in such domains clearly did not affect the housekeeping functions of the proteins, and in fact, they were favorably selected in evolution during exposure to increasingly severe nutrient deprivation. In such a gradually changing environment, the network of the interactome, therefore, increased in size and complexity in punctuated steps, reaching equilibrium at each step. This was also suggested previously for the evolution of thermotolerant organisms, selected during global warming [153]. Of note, there are several other non-nucleic acid nucleotides that are diverse in synthesis, structure, and function. Major examples include: ppApp, which is the adenine counterpart of (p)ppGpp; cyclic nucleotides, such as cAMP, the first recognized nucleotide second messenger; the composite cyclic-GMP-AMP or cGAMP, made from GTP and ATP. Each second messenger acts as a ligand to activate its own interactome of proteins that regulate pathway-specific signaling cascades, triggering specific physiological effects.