Orchestrating cellular responses: A molecular symphony by the alarmones (p)ppGpp and the mysterious Ap4A

The stringent response, mediated by the bacterial alarmones ppGpp and pppGpp (collectively known as (p)ppGpp), regulates diverse cellular processes, including protein trafficking via the signal recognition particle (SRP) pathway. Additionally, diadenosine tetraphosphate (Ap4A) has been emerging as a critical nucleotide with roles in stress responses. The primary objective of this dissertation was to investigate whether the (p)ppGpp-mediated regulation of bacterial SRP is conserved in chloroplasts and to explore the interplay between (p)ppGpp and Ap4A in bacteria. Biochemical and structural analyses revealed that (p)ppGpp binds to the GTPase-containing NG domains of the chloroplast SRP receptor (cpFtsY-NG) and its SRP54 subunit, preventing their GTP-dependent association. Structural characterization of the cpFtsY-NG:ppGpp complex showed that ppGpp mimics GDP binding but inhibits cpFtsY:cpSRP54 complex formation via its pyrophosphate moiety, disrupting mutual GTPase stimulation. These findings provide the first description of (p)ppGpp regulation in the chloroplast SRP pathway and suggest a broader role in plastid stress responses. In parallel, I investigated Ap4A as a potential regulatory nucleotide in bacteria and humans. My data demonstrates that heat stress triggers Ap4A accumulation in E. coli and human cells, suggesting a conserved mechanism extending across domains of life. Using a reconstituted cell-free system, I confirmed that Ap4A accumulation is temperature-dependent and independent of an active translational apparatus. Moreover, Ap4A was shown to bind to B. subtilis DarB with a dissociation constant of 3.78 µM, as determined by isothermal titration calorimetry (ITC). I could solidify this finding by additional structural insights provided by successful crystallographic determination of the DarB:Ap4A complex at 2.5 Å resolution. Strikingly, Ap4A interfered with the DarB-mediated enhancement of RelNTD enzymatic activity, suppressing (p)ppGpp synthesis similarly to c-di-AMP. Furthermore, DL-serine hydroxamate (SHX)-induced E. coli cultures exhibited synchronized peaks of Ap4A and ppGpp, supporting a potential co-regulation mechanism. These findings suggest that Ap4A and (p)ppGpp may operate within a shared regulatory network to mediate stress responses in bacteria. Altogether, this work advances our understanding of nucleotide signaling in bacteria, plants, and humans, revealing conserved regulatory roles for (p)ppGpp and Ap4A in stress adaptation and highlighting their potential implications in cellular and environmental resilience across domains of life.