Browse
Submit Data
Databases
API
Help

Dataset Information

15 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

The RelA hydrolase domain acts as a molecular switch for (p)ppGpp synthesis.

ABSTRACT: Bacteria synthesize guanosine tetra- and penta phosphate (commonly referred to as (p)ppGpp) in response to environmental stresses. (p)ppGpp reprograms cell physiology and is essential for stress survival, virulence and antibiotic tolerance. Proteins of the RSH superfamily (RelA/SpoT Homologues) are ubiquitously distributed and hydrolyze or synthesize (p)ppGpp. Structural studies have suggested that the shift between hydrolysis and synthesis is governed by conformational antagonism between the two active sites in RSHs. RelA proteins of γ-proteobacteria exclusively synthesize (p)ppGpp and encode an inactive pseudo-hydrolase domain. Escherichia coli RelA synthesizes (p)ppGpp in response to amino acid starvation with cognate uncharged tRNA at the ribosomal A-site, however, mechanistic details to the regulation of the enzymatic activity remain elusive. Here, we show a role of the enzymatically inactive hydrolase domain in modulating the activity of the synthetase domain of RelA. Using mutagenesis screening and functional studies, we identify a loop region (residues 114-130) in the hydrolase domain, which controls the synthetase activity. We show that a synthetase-inactive loop mutant of RelA is not affected for tRNA binding, but binds the ribosome less efficiently than wild type RelA. Our data support the model that the hydrolase domain acts as a molecular switch to regulate the synthetase activity.

SUBMITTER: Sinha AK

PROVIDER: S-EPMC8012599 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

The RelA hydrolase domain acts as a molecular switch for (p)ppGpp synthesis

Project description:Bacteria synthesize guanosine tetra- and penta phosphate (commonly referred to as (p)ppGpp) in response to environmental stresses. (p)ppGpp reprograms cell physiology and is essential for stress survival, virulence and antibiotic tolerance. Proteins of the RSH superfamily (RelA/SpoT Homologues) are ubiquitously distributed and hydrolyze or synthesize (p)ppGpp. Structural studies have suggested that the shift between hydrolysis and synthesis is governed by conformational antagonism between the two active sites in RSHs. RelA proteins of γ-proteobacteria exclusively synthesize (p)ppGpp and encode an inactive pseudo-hydrolase domain. Escherichia coli RelA synthesizes (p)ppGpp in response to amino acid starvation with cognate uncharged tRNA at the ribosomal A-site, however, mechanistic details to the regulation of the enzymatic activity on the ribosome remain elusive. Here, we show a novel role of the enzymatically inactive hydrolase domain in modulating the activity of the synthetase domain of RelA. Using random mutagenesis screening and functional studies, we identify a loop region (residues 114-130) in the hydrolase domain, which controls the synthetase activity. We show that a synthetase-inactive loop mutant of RelA is not affected for tRNA binding, but binds the ribosome less efficiently than wildtype RelA. Our data provide strong evidence to support the model that the hydrolase domain acts as a molecular switch to regulate the synthetase activity.

2021-03-31 | GSE150416 | GEO

The RelA hydrolase domain acts as a molecular switch for (p)ppGpp synthesis

Project description:The RelA hydrolase domain acts as a molecular switch for (p)ppGpp synthesis

| PRJNA632006 | ENA

Molecular mutagenesis of ppGpp: turning a RelA activator into an inhibitor.

Project description:The alarmone nucleotide (p)ppGpp is a key regulator of bacterial metabolism, growth, stress tolerance and virulence, making (p)ppGpp-mediated signaling a promising target for development of antibacterials. Although ppGpp itself is an activator of the ribosome-associated ppGpp synthetase RelA, several ppGpp mimics have been developed as RelA inhibitors. However promising, the currently available ppGpp mimics are relatively inefficient, with IC50 in the sub-mM range. In an attempt to identify a potent and specific inhibitor of RelA capable of abrogating (p)ppGpp production in live bacterial cells, we have tested a targeted nucleotide library using a biochemical test system comprised of purified Escherichia coli components. While none of the compounds fulfilled this aim, the screen has yielded several potentially useful molecular tools for biochemical and structural work.

| S-EPMC5291098 | biostudies-literature

Regulation of the opposing (p)ppGpp synthetase and hydrolase activities in a bifunctional RelA/SpoT homologue from Staphylococcus aureus.

Project description:The stringent response is characterized by (p)ppGpp synthesis resulting in repression of translation and reprogramming of the transcriptome. In Staphylococcus aureus, (p)ppGpp is synthesized by the long RSH (RelA/SpoT homolog) enzyme, RelSau or by one of the two short synthetases (RelP, RelQ). RSH enzymes are characterized by an N-terminal enzymatic domain bearing distinct motifs for (p)ppGpp synthetase or hydrolase activity and a C-terminal regulatory domain (CTD) containing conserved motifs (TGS, DC and ACT). The intramolecular switch between synthetase and hydrolase activity of RelSau is crucial for the adaption of S. aureus to stress (stringent) or non-stress (relaxed) conditions. We elucidated the role of the CTD in the enzymatic activities of RelSau. Growth pattern, transcriptional analyses and in vitro assays yielded the following results: i) in vivo, under relaxed conditions, as well as in vitro, the CTD inhibits synthetase activity but is not required for hydrolase activity; ii) under stringent conditions, the CTD is essential for (p)ppGpp synthesis; iii) RelSau lacking the CTD exhibits net hydrolase activity when expressed in S. aureus but net (p)ppGpp synthetase activity when expressed in E. coli; iv) the TGS and DC motifs within the CTD are required for correct stringent response, whereas the ACT motif is dispensable, v) Co-immunoprecipitation indicated that the CTD interacts with the ribosome, which is largely dependent on the TGS motif. In conclusion, RelSau primarily exists in a synthetase-OFF/hydrolase-ON state, the TGS motif within the CTD is required to activate (p)ppGpp synthesis under stringent conditions.

| S-EPMC6053245 | biostudies-literature

The stringent factor RelA adopts an open conformation on the ribosome to stimulate ppGpp synthesis.

Project description:Under stress conditions, such as nutrient starvation, deacylated tRNAs bound within the ribosomal A-site are recognized by the stringent factor RelA, which converts ATP and GTP/GDP to (p)ppGpp. The signaling molecules (p)ppGpp globally rewire the cellular transcriptional program and general metabolism, leading to stress adaptation. Despite the additional importance of the stringent response for regulation of bacterial virulence, antibiotic resistance and persistence, structural insight into how the ribosome and deacylated-tRNA stimulate RelA-mediated (p)ppGpp has been lacking. Here, we present a cryo-EM structure of RelA in complex with the Escherichia coli 70S ribosome with an average resolution of 3.7 Å and local resolution of 4 to >10 Å for RelA. The structure reveals that RelA adopts a unique 'open' conformation, where the C-terminal domain (CTD) is intertwined around an A/T-like tRNA within the intersubunit cavity of the ribosome and the N-terminal domain (NTD) extends into the solvent. We propose that the open conformation of RelA on the ribosome relieves the autoinhibitory effect of the CTD on the NTD, thus leading to stimulation of (p)ppGpp synthesis by RelA.

| S-EPMC5291266 | biostudies-literature

HipA-triggered growth arrest and ?-lactam tolerance in Escherichia coli are mediated by RelA-dependent ppGpp synthesis.

Project description:Persistence is a phenomenon whereby a subpopulation of bacterial cells enters a transient growth-arrested state that confers antibiotic tolerance. While entrance into persistence has been linked to the activities of toxin proteins, the molecular mechanisms by which toxins induce growth arrest and the persistent state remain unclear. Here, we show that overexpression of the protein kinase HipA in Escherichia coli triggers growth arrest by activating synthesis of the alarmone guanosine tetraphosphate (ppGpp) by the enzyme RelA, a signal typically associated with amino acid starvation. We further demonstrate that chemically suppressing ppGpp synthesis with chloramphenicol relieves inhibition of DNA replication initiation and RNA synthesis in HipA-arrested cells and restores vulnerability to ?-lactam antibiotics. HipA-arrested cells maintain glucose uptake and oxygen consumption and accumulate amino acids as a consequence of translational inhibition. We harness the active metabolism of HipA-arrested cells to provide a bacteriophage-resistant platform for the production of biotechnologically relevant compounds, which may represent an innovative solution to the costly problem of phage contamination in industrial fermentations.

| S-EPMC3697643 | biostudies-literature

Arabidopsis RelA/SpoT homologs implicate (p)ppGpp in plant signaling.

Project description:Arabidopsis RPP5 is a member of a large class of pathogen resistance genes encoding nucleotide-binding sites and leucine-rich repeat domains. Yeast two-hybrid analysis showed that RPP5 specifically interacts with At-RSH1, an Arabidopsis RelA/SpoT homolog. In Escherichia coli, RelA and SpoT determine the level of guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp), which are the effector nucleotides of the bacterial stringent response. Functional analysis in E. coli and in Streptomyces coelicolor A3 (2) showed that At-RSH1 confers phenotypes associated with (p)ppGpp synthesis. We characterized two additional Arabidopsis RelA/SpoT homologs, At-RSH2 and At-RSH3. At-RSH genes may regulate a rapid plant (p)ppGpp-mediated response to pathogens and other stresses.

| S-EPMC16311 | biostudies-literature

Vemurafenib acts as molecular switch in Langerhans Cell Histiocytosis

Project description:Langerhans cell histiocytosis (LCH) is a neoplasm marked by the accumulation of CD1a+CD207+ cells. It is most commonly driven by a somatic activating mutation in the BRAF kinase (BRAFV600E). Multisystem disease with risk-organ involvement requires intensive chemotherapy, making BRAF-inhibitors an attractive option. Here, we present a comprehensive analysis of the course of an LCH patient treated with the combination of vemurafenib with salvage chemotherapy who achieved complete clinical and molecular remission. We conclusively show that there is no relationship between blood-BRAF levels and clinical presentation during treatment with vemurafenib, but that vemurafenib leads to a fast, efficient, but reversible inhibition of clinical manifestations of high-risk LCH such as cytopenia, fever, and hypoproteinemia. In line, serum levels of inflammatory cytokines exactly mirror vemurafenib administration. Genotyping for the BRAFV600E mutation shows its presence in multiple hematopoietic cell types, including NK cells and granulocytes. Single-cell transcriptome analysis of peripheral blood and bone marrow cells at time of diagnosis and during treatment indicate that RAF-inhibition abrogates the expression of inflammatory cytokines previously implicated in LCH. Together, our data suggest that while the CD1a+CD207+ histiocytes are the hallmark of LCH, other BRAF-mutated cells themselves contribute significantly to morbidity in patients with multisystem LCH.

2021-09-23 | GSE175480 | GEO

The AAA3 domain of cytoplasmic dynein acts as a switch to facilitate microtubule release.

Project description:Cytoplasmic dynein is an AAA+ motor responsible for intracellular cargo transport and force generation along microtubules (MTs). Unlike kinesin and myosin, dynein contains multiple ATPase subunits, with AAA1 serving as the primary catalytic site. ATPase activity at AAA3 is also essential for robust motility, but its role in dynein's mechanochemical cycle remains unclear. Here, we introduced transient pauses in Saccharomyces cerevisiae dynein motility by using a slowly hydrolyzing ATP analog. Analysis of pausing behavior revealed that AAA3 hydrolyzes nucleotide an order of magnitude more slowly than AAA1, and the two sites do not coordinate. ATPase mutations to AAA3 abolish the ability of dynein to modulate MT release. Nucleotide hydrolysis at AAA3 lifts this 'MT gate' to allow fast motility. These results suggest that AAA3 acts as a switch that repurposes cytoplasmic dynein for fast cargo transport and MT-anchoring tasks in cells.

| S-EPMC4286497 | biostudies-literature

RelB acts as a molecular switch driving chronic inflammation in glioblastoma multiforme.

Project description:Glioblastoma multiforme (GBM) is a primary brain tumor characterized by extensive necrosis and immunosuppressive inflammation. The mechanisms by which this inflammation develops and persists in GBM remain elusive. We identified two cytokines interleukin-1? (IL-1) and oncostatin M (OSM) that strongly negatively correlate with patient survival. We found that these cytokines activate RelB/p50 complexes by a canonical NF-?B pathway, which surprisingly drives expression of proinflammatory cytokines in GBM cells, but leads to their inhibition in non-transformed astrocytes. We discovered that one allele of the gene encoding deacetylase Sirtuin 1 (SIRT1), needed for repression of cytokine genes, is deleted in 80% of GBM tumors. Furthermore, RelB specifically interacts with a transcription factor Yin Yang 1 (YY1) in GBM cells and activates GBM-specific gene expression programs. As a result, GBM cells continuously secrete proinflammatory cytokines and factors attracting/activating glioma-associated microglia/macrophages and thus, promote a feedforward inflammatory loop.

| S-EPMC6541631 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data