RelA is a long RelA/SpoT homolog (RSH) enzyme and the major (p)ppGpp synthetase (GTP pyrophosphokinase, ppGpp synthase I) that mediates the bacterial stringent response. It catalyzes the transfer of the beta,gamma-pyrophosphate group of ATP to the 3'-hydroxyl of GDP or GTP, producing the alarmones ppGpp or pppGpp plus AMP. The enzyme is a multidomain protein comprising an N-terminal catalytic region (a nucleotidyltransferase/synthetase domain together with a degenerate, largely inactive HD/hydrolase domain) and C-terminal regulatory modules (TGS and ACT domains) that couple catalysis to the cell's nutritional and translational state. RelA is a cytosolic, ribosome-associated enzyme; it is activated during amino-acid starvation when deacylated (uncharged) tRNA enters the ribosomal A site of a stalled ribosome, stimulating (p)ppGpp synthesis. The resulting (p)ppGpp acts as a global second messenger that binds RNA polymerase (with the cofactor DksA) and other targets to reprogram transcription and translation, downregulate ribosome biogenesis and de novo purine/nucleotide biosynthesis, and shift cells from growth toward survival under nutrient limitation. In Pseudomonas putida, (p)ppGpp produced by RelA (together with the bifunctional SpoT) also feeds into starvation-induced biofilm dispersal and carbon-storage (polyhydroxyalkanoate) physiology.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0005886 plasma membrane | IEA GO_REF:0000118 | MARK AS OVER ANNOTATED | Summary: RelA is a cytosolic, ribosome-associated enzyme, not an integral membrane protein; there is no synthetase or family-level basis for plasma membrane localization. Reason: This PANTHER/TreeGrafter IEA over-propagates a plasma membrane location to RelA. The protein has no transmembrane segments or membrane-targeting features, and the literature consensus places long RSH RelA enzymes in the cytosol associated with translating/stalled ribosomes (the mechanistically relevant compartment for ribosome-dependent activation). A more appropriate cellular component, if any, would be cytoplasm. |
| GO:0008728 GTP diphosphokinase activity | IEA GO_REF:0000118 | ACCEPT | Summary: Core molecular function: RelA is a GTP pyrophosphokinase / (p)ppGpp synthetase that transfers pyrophosphate from ATP to GDP/GTP to form (p)ppGpp. Reason: This matches the well-established and UniProt-supported primary activity of RelA (ppGpp synthase I) and the conserved synthetase domain of the RelA/SpoT family. Represents a core function of the gene. |
| GO:0008893 guanosine-3',5'-bis(diphosphate) 3'-diphosphatase activity | IEA GO_REF:0000118 | MARK AS OVER ANNOTATED | Summary: This is the (p)ppGpp hydrolase activity. In canonical long RelA proteins the N-terminal HD/hydrolase domain is degenerate and largely inactive; ppGpp hydrolysis is provided by the bifunctional paralog SpoT, so this is most likely an over-annotation propagated from the bifunctional branch of the family tree. Reason: RelA in gamma-proteobacteria (the E. coli/P. putida paradigm) functions primarily as a synthetase; the HD motif required for hydrolase chemistry is typically degenerate. The TreeGrafter inference cannot distinguish RelA from bifunctional SpoT/Rel within the RSH family. The hydrolase activity is not experimentally established for P. putida RelA and is biologically attributed to SpoT. Flagging as over-annotated rather than REMOVE because residual HD sequence exists and the activity cannot be formally excluded without assay. |
| GO:0015949 nucleobase-containing small molecule interconversion | IEA GO_REF:0000117 | MODIFY | Summary: Overly general biological process term; the specific process catalyzed is guanosine tetraphosphate (ppGpp) biosynthesis. Reason: The ARBA inference is not wrong but is uninformatively broad. The precise process is (p)ppGpp biosynthesis, which is better captured by the more specific child term, consistent with the GTP diphosphokinase activity and the guanosine tetraphosphate metabolic process annotations. Proposed replacements: guanosine tetraphosphate biosynthetic process |
| GO:0015969 guanosine tetraphosphate metabolic process | IEA GO_REF:0000120 | ACCEPT | Summary: Accurate biological process for RelA, whose synthetase activity produces the alarmone ppGpp (guanosine tetraphosphate). Reason: Directly supported by the synthetase function and family assignment; represents a core biological process of the gene. A more specific biosynthetic child term is also applicable (see GO:0015970 proposed above). |
| GO:0042594 response to starvation | IEA GO_REF:0000118 | ACCEPT | Summary: RelA mediates the stringent response, producing (p)ppGpp upon amino-acid (and broader nutrient) starvation; this process annotation is appropriate. Reason: Consistent with the canonical role of RelA in the stringent response and supported in P. putida KT2440 by starvation/(p)ppGpp studies. A more precise term such as cellular response to starvation could be considered, but the current term is acceptable and informative. |
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Download this section (compressed HTML)Q: Is the degenerate HD/hydrolase domain of P. putida RelA catalytically inactive in vivo, or does RelA retain measurable (p)ppGpp hydrolase activity as some long RSH proteins do?
Q: Under which nutrient-limitation conditions in P. putida is RelA (versus SpoT) the dominant (p)ppGpp source?
Experiment: In vitro biochemical assay of purified P. putida RelA to measure (p)ppGpp synthetase activity (ATP + GDP/GTP) and to test for residual 3'-pyrophosphohydrolase activity, with and without ribosome/deacylated-tRNA stimulation.
Experiment: Quantify (p)ppGpp accumulation kinetics in wild-type, delta relA, delta spoT, and ppGpp0 (delta relA delta spoT) P. putida strains under amino-acid starvation to define RelA's specific contribution.
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