RelA-SpoT Homolog toxins pyrophosphorylate the CCA end of tRNA to inhibit protein synthesis.

RelA-SpoT Homolog (RSH) enzymes control bacterial physiology through synthesis and degradation of the nucleotide alarmone (p)ppGpp. We recently discovered multiple families of small alarmone synthetase (SAS) RSH acting as toxins of toxin-antitoxin (TA) modules, with the FaRel subfamily of toxSAS abrogating bacterial growth by producing an analog of (p)ppGpp, (pp)pApp. Here we probe the mechanism of growth arrest used by four experimentally unexplored subfamilies of toxSAS: FaRel2, PhRel, PhRel2, and CapRel. Surprisingly, all these toxins specifically inhibit protein synthesis. To do so, they transfer a pyrophosphate moiety from ATP to the tRNA 3' CCA. The modification inhibits both tRNA aminoacylation and the sensing of cellular amino acid starvation by the ribosome-associated RSH RelA. Conversely, we show that some small alarmone hydrolase (SAH) RSH enzymes can reverse the pyrophosphorylation of tRNA to counter the growth inhibition by toxSAS. Collectively, we establish RSHs as RNA-modifying enzymes.

(p)ppGpp controls stringent factors by exploiting antagonistic allosteric coupling between catalytic domains.

Amino acid starvation is sensed by Escherichia coli RelA and Bacillus subtilis Rel through monitoring the aminoacylation status of ribosomal A-site tRNA. These enzymes are positively regulated by their product-the alarmone nucleotide (p)ppGpp-through an unknown mechanism. The (p)ppGpp-synthetic activity of Rel/RelA is controlled via auto-inhibition by the hydrolase/pseudo-hydrolase (HD/pseudo-HD) domain within the enzymatic N-terminal domain region (NTD). We localize the allosteric pppGpp site to the interface between the SYNTH and pseudo-HD/HD domains, with the alarmone stimulating Rel/RelA by exploiting intra-NTD autoinhibition dynamics. We show that without stimulation by pppGpp, starved ribosomes cannot efficiently activate Rel/RelA. Compromised activation by pppGpp ablates Rel/RelA function in vivo, suggesting that regulation by the second messenger (p)ppGpp is necessary for mounting an acute starvation response via coordinated enzymatic activity of individual Rel/RelA molecules. Control by (p)ppGpp is lacking in the E. coli (p)ppGpp synthetase SpoT, thus explaining its weak synthetase activity.

Regulation of ppGpp Synthesis and Its Impact on Chloroplast Biogenesis during Early Leaf Development in Rice.

Guanosine tetraphosphate (ppGpp) is known as an alarmone that mediates bacterial stress responses. In plants, ppGpp is synthesized in chloroplasts from GTP and ATP and functions as a regulator of chloroplast gene expression to affect photosynthesis and plant growth. This observation indicates that ppGpp metabolism is closely related to chloroplast function, but the regulation of ppGpp and its role in chloroplast differentiation are not well understood. In rice, ppGpp directly inhibits plastidial guanylate kinase (GKpm), a key enzyme in GTP biosynthesis. GKpm is highly expressed during early leaf development in rice, and the GKpm-deficient mutant, virescent-2 (v2), develops chloroplast-deficient chlorotic leaves under low-temperature conditions. To examine the relationship between GTP synthesis and ppGpp homeostasis, we generated transgenic rice plants over-expressing RSH3, a protein known to act as a ppGpp synthase. When RSH3 was overexpressed in v2, the leaf chlorosis was more severe. Although the RSH3 overexpression in the wild type caused no visible effects, pulse amplitude modulation fluorometer measurements indicated that photosynthetic rates were reduced in this line. This finding implies that the regulation of ppGpp synthesis in rice is involved in the maintenance of the GTP pool required to regulate plastid gene expression during early chloroplast biogenesis. We further investigated changes in the expressions of RelA/SpoT Homolog (RSH) genes encoding ppGpp synthases and hydrolases during the same period. Comparing the expression of these genes with the cellular ppGpp content suggests that the basal ppGpp level is determined by the antagonistic action of multiple RSH enzymatic activities during early leaf development in rice.

Regulation of stringent factor by branched-chain amino acids.

When faced with amino acid starvation, prokaryotic cells induce a stringent response that modulates their physiology. The stringent response is manifested by production of signaling molecules guanosine 5'-diphosphate,3'-diphosphate (ppGpp) and guanosine 5'-triphosphate,3'-diphosphate (pppGpp) that are also called alarmones. In many species, alarmone levels are regulated by a multidomain bifunctional alarmone synthetase/hydrolase called Rel. In this enzyme, there is an ACT domain at the carboxyl region that has an unknown function; however, similar ACT domains are present in other enzymes that have roles in controlling amino acid metabolism. In many cases, these other ACT domains have been shown to allosterically regulate enzyme activity through the binding of amino acids. Here, we show that the ACT domain present in the Rhodobacter capsulatus Rel alarmone synthetase/hydrolase binds branched-chain amino acids valine and isoleucine. We further show that the binding of these amino acids stimulates alarmone hydrolase activity both in vitro and in vivo. Furthermore, we found that the ACT domain present in Rel proteins from many diverse species also binds branched-chain amino acids. These results indicate that the cellular concentration of amino acids can directly affect Rel alarmone synthetase/hydrolase activity, thus adding another layer of control to current models of cellular control of the stringent response.

In Silico Study of the RSH (RelA/SpoT Homologs) Gene Family and Expression Analysis in Response to PGPR Bacteria and Salinity in Brassica napus.

Among several mechanisms involved in the plant stress response, synthesis of guanosine tetra and pentaphosphates (alarmones), homologous to the bacterial stringent response, is of crucial importance. Plant alarmones affect, among others, photosynthetic activity, metabolite accumulation, and nutrient remobilization, and thus regulate plant growth and development. The plant RSH (RelA/SpoT homolog) genes, that encode synthetases and/or hydrolases of alarmones, have been characterized in a limited number of plant species, e.g., Arabidopsis thaliana, Oryza sativa, and Ipomoea nil. Here, we used dry-to-wet laboratory research approaches to characterize RSH family genes in the polyploid plant Brassica napus. There are 12 RSH genes in the genome of rapeseed that belong to four types of RSH genes: 6 RSH1, 2 RSH2, 3 RSH3, and 1 CRSH. BnRSH genes contain 13-24 introns in RSH1, 2-6 introns in RSH2, 1-6 introns in RSH3, and 2-3 introns in the CRSH genes. In the promoter regions of the RSH genes, we showed the presence of regulatory elements of the response to light, plant hormones, plant development, and abiotic and biotic stresses. The wet-lab analysis showed that expression of BnRSH genes is generally not significantly affected by salt stress, but that the presence of PGPR bacteria, mostly of Serratia sp., increased the expression of BnRSH significantly. The obtained results show that BnRSH genes are differently affected by biotic and abiotic factors, which indicates their different functions in plants.

The checkpoint kinase TOR (target of rapamycin) regulates expression of a nuclear-encoded chloroplast RelA-SpoT homolog (RSH) and modulates chloroplast ribosomal RNA synthesis in a unicellular red alga.

Chloroplasts are plant organelles that carry out oxygenic photosynthesis. Chloroplast biogenesis depends upon chloroplast ribosomes and their translational activity. However, regulation of chloroplast ribosome biogenesis remains an important unanswered question. In this study, we found that inhibition of target of rapamycin (TOR), a general eukaryotic checkpoint kinase, results in a decline in chloroplast ribosomal RNA (rRNA) transcription in the unicellular red alga, Cyanidioschyzon merolae. Upon TOR inhibition, transcriptomics and other analyses revealed increased expression of a nuclear-encoded chloroplast RelA-SpoT homolog (RSH) gene (CmRSH4b), which encodes a homolog of the guanosine 3'-diphosphate 5'-diphosphate (ppGpp) synthetases that modulate rRNA synthesis in bacteria. Using an Escherichia coli mutant lacking ppGpp, CmRSH4b was demonstrated to have ppGpp synthetase activity. Expression analysis of a green fluorescent protein-fused protein indicated that CmRSH4b localizes to the chloroplast, and overexpression of the CmRSH4b gene resulted in a decrease of chloroplast rRNA synthesis concomitant with growth inhibition and reduction of chloroplast size. Biochemical analyses using C. merolae cell lysates or purified recombinant proteins revealed that ppGpp inhibits bacteria-type RNA polymerase-dependent chloroplast rRNA synthesis as well as a chloroplast guanylate kinase. These results suggest that CmRSH4b-dependent ppGpp synthesis in chloroplasts is an important regulator of chloroplast rRNA transcription. Nuclear and mitochondrial rRNA transcription were both reduced by TOR inhibition, suggesting that the biogeneses of the three independent ribosome systems are interconnected by TOR in plant cells.

Phylogenetic analysis of proteins involved in the stringent response in plant cells.

The nucleotide (p)ppGpp is a second messenger that controls the stringent response in bacteria. The stringent response modifies expression of a large number of genes and metabolic processes and allows bacteria to survive under fluctuating environmental conditions. Recent genome sequencing analyses have revealed that genes responsible for the stringent response are also found in plants. These include (p)ppGpp synthases and hydrolases, RelA/SpoT homologs (RSHs), and the pppGpp-specific phosphatase GppA/Ppx. However, phylogenetic relationship between enzymes involved in bacterial and plant stringent responses is as yet generally unclear. Here, we investigated the origin and evolution of genes involved in the stringent response in plants. Phylogenetic analysis and primary structures of RSH homologs from different plant phyla (including Embryophyta, Charophyta, Chlorophyta, Rhodophyta and Glaucophyta) indicate that RSH gene families were introduced into plant cells by at least two independent lateral gene transfers from the bacterial Deinococcus-Thermus phylum and an unidentified bacterial phylum; alternatively, they were introduced into a proto-plant cell by a lateral gene transfer from the endosymbiotic cyanobacterium followed by gene loss of an ancestral RSH gene in the cyanobacterial linage. Phylogenetic analysis of gppA/ppx families indicated that plant gppA/ppx homologs form an individual cluster in the phylogenetic tree, and show a sister relationship with some bacterial gppA/ppx homologs. Although RSHs contain a plastidial transit peptide at the N terminus, GppA/Ppx homologs do not, suggesting that plant GppA/Ppx homologs function in the cytosol. These results reveal that a proto-plant cell obtained genes for the stringent response by lateral gene transfer events from different bacterial phyla and have utilized them to control metabolism in plastids and the cytosol.

Co-crystalization reveals the interaction between AtYchF1 and ppGpp.

AtYchF1 is an unconventional G-protein in Arabidopsis thaliana that exhibits relaxed nucleotide-binding specificity. The bindings between AtYchF1 and biomolecules including GTP, ATP, and 26S rRNA have been reported. In this study, we demonstrated the binding of AtYchF1 to ppGpp in addition to the above molecules. AtYchF1 is a cytosolic protein previously reported as a negative regulator of both biotic and abiotic stresses while the accumulation of ppGpp in the cytoplasm induces retarded plant growth and development. By co-crystallization, in vitro pull-down experiments, and hydrolytic biochemical assays, we demonstrated the binding and hydrolysis of ppGpp by AtYchF1. ppGpp inhibits the binding of AtYchF1 to ATP, GTP, and 26S rRNA. The ppGpp hydrolyzing activity of AtYchF1 failed to be activated by AtGAP1. The AtYchF1-ppGpp co-crystal structure suggests that ppGpp might prevent His136 from executing nucleotide hydrolysis. In addition, upon the binding of ppGpp, the conformation between the TGS and helical domains of AtYchF1 changes. Such structural changes probably influence the binding between AtYchF1 and other molecules such as 26S rRNA. Since YchF proteins are conserved among different kingdoms of life, the findings advance the knowledge on the role of AtYchF1 in regulating nucleotide signaling as well as hint at the possible involvement of YchF proteins in regulating ppGpp level in other species.

Identification and characterization of the Ipomoea nil RelA/SpoT Homologs (InRSHs) and potential directions of their transcriptional regulation.

Although the stringent response has been known for more than half a century and has been well studied in bacteria, only the research of the past 19 years revealed that the homologous mechanism is conserved in plants. The plant RelA/SpoT Homolog (RSH) genes have been identified and characterized in a limited number of plant species, whereas products of their catalytic activities, (p)ppGpp (alarmones), have been shown to accumulate mainly in chloroplasts. Here, we identified full-length sequences of the Ipomoea nil RSH genes (InRSH1, InRSH2 and InCRSH), determined their copy number in the I. nil genome as well as the structural conservancy between InRSHs and their Arabidopsis and rice orthologs. We showed that InRSHs are differentially expressed in I. nil organ tissues and that only InRSH2 is upregulated in response to salt, osmotic and drought stress. Our results of the E. coli relA/spoT mutant complementation test suggest that InRSH1 is likely a (p)ppGpp hydrolase, InCRSH - synthetase and InRSH2 shows both activities. Finally, we referred our results to the recently published I. nil genomic and proteomic data and uncovered the complexity of the I. nil RSH family as well as potential ways of the InRSH transcriptional regulation.

Identification and Functional Analysis of RelA/SpoT Homolog (RSH) Genes in Deinococcus radiodurans.

To identify the global effects of (p)ppGpp in the gram-positive bacterium Deinococcus radiodurans, which exhibits remarkable resistance to radiation and other stresses, RelA/SpoT homolog (RSHs) mutants were constructed by direct deletion mutagenesis. The results showed that RelA has both synthesis and hydrolysis domains of (p)ppGpp, whereas RelQ only synthesizes (p)ppGpp in D. radiodurans. The growth assay for mutants and complementation analysis revealed that deletion of relA and relQ sensitized the cells to H2O2, heat shock, and amino acid limitation. Comparative proteomic analysis revealed that the bifunctional RelA is involved in DNA repair, molecular chaperone functions, transcription, the tricarboxylic acid cycle, and metabolism, suggesting that relA maintains the cellular (p)ppGpp levels and plays a crucial role in oxidative resistance in D. radiodurans. The D. radiodurans relA and relQ genes are responsible for (p)ppGpp synthesis/hydrolysis and (p)ppGpp hydrolysis, respectively. (p)ppGpp integrates a general stress response with a targeted re-programming of gene regulation to allow bacteria to respond appropriately towards heat shock, oxidative stress, and starvation. This is the first identification of RelA and RelQ involvement in response to oxidative, heat shock, and starvation stresses in D. radiodurans, which further elucidates the remarkable resistance of this bacterium to stresses.

AtObgC-AtRSH1 interaction may play a vital role in stress response signal transduction in Arabidopsis.

The interaction of Obg (Spo0B-associated GTP-binding protein) GTPase and SpoT, which is a bifunctional ppGpp (guanosine 3',5'-bispyrophosphate) hydrolase/synthetase, is vital for the modulation of intracellular ppGpp levels during bacterial responses to environmental cues. It has been recently reported that the ppGpp level is also inducible by various stresses in the chloroplasts of plant cells. However, the function of the Obg-SpoT interaction in plants remains elusive. The results from the present and previous studies suggest that AtRSH1 is a putative bacterial SpoT homolog in Arabidopsis and that its transcription levels are responsive to wounding and salt stresses. In this study, we used a yeast two-hybrid analysis to map the regions required for the AtObgC-AtRSH1 interaction. Moreover, protein-protein docking simulations revealed reasonable geometric and electrostatic complementarity in the binding surfaces of the two proteins. The data support our experimental results, which suggest that the conserved domains in AtObgC and the N terminus of AtRSH1 containing the TGS domain contribute to their interaction. In addition, quantitative real-time PCR (qRT-PCR) analyses showed that the expression of AtObgC and AtRSH1 exhibit a similar inhibition pattern under wounding and salt-stress conditions, but the inhibition pattern was not greatly influenced by the presence or absence of light. Based on in vivo analyses, we further confirmed that the AtRSH1 and AtObgC proteins similarly localize in chloroplasts. Based on these results, we propose that the AtObgC-AtRSH1 interaction plays a vital role in ppGpp-mediated stress responses in chloroplasts.