A Primase-Induced Conformational Switch Controls the Stability of the Bacterial Replisome.
Ontology highlight
ABSTRACT:
SUBMITTER: Monachino E
PROVIDER: S-EPMC7335327 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:Histone H3 lysine 9 methylation (H3K9me) mediates heterochromatic gene silencing and is important for genome stability and the regulation of gene expression. The establishment and epigenetic maintenance of heterochromatin involve the recruitment of H3K9 methyltransferases to specific sites on DNA, followed by the recognition of pre-existing H3K9me by the methyltransferase and methylation of proximal histone H3. This positive feedback loop must be tightly regulated to prevent deleterious epigenetic gene silencing. Extrinsic anti-silencing mechanisms involving histone demethylation or boundary elements help limit the spread of inappropriate H3K9me. However, how H3K9 methyltransferase activity is locally restricted or prevented from initiating random H3K9me—which would lead to aberrant gene silencing and epigenetic instability—is not fully understood. Here we reveal an autoinhibited conformation in the conserved H3K9 methyltransferase Clr4 (Suv39h) of the fission yeast Schizosaccharomyces pombe that has a critical role in preventing aberrant heterochromatin formation. Biochemical and X-ray crystallographic data show that an internal loop in Clr4 inhibits the catalytic activity of this enzyme by blocking the histone H3K9 substrate-binding pocket, and that automethylation of specific lysines in this loop promotes a conformational switch that enhances the H3K9me activity of Clr4. Mutations that are predicted to disrupt this regulation lead to aberrant H3K9me, loss of heterochromatin domains, and growth inhibition, demonstrating the importance of the intrinsic inhibition and auto-activation of Clr4 in regulating the deposition of H3K9me and in preventing epigenetic instability. Together, conservation of the Clr4 autoregulatory loop in other H3K9 methyltransferases and the automethylation of a corresponding lysine in the human SUV39H2 homologue suggest that the mechanism described here is broadly conserved.
2018-07-17 | GSE102905 | GEO
Project description:Histone H3 lysine 9 methylation (H3K9me) mediates heterochromatic gene silencing and is important for genome stability and the regulation of gene expression1-4. The establishment and epigenetic maintenance of heterochromatin involve the recruitment of H3K9 methyltransferases to specific sites on DNA, followed by the recognition of pre-existing H3K9me by the methyltransferase and methylation of proximal histone H35-11. This positive feedback loop must be tightly regulated to prevent deleterious epigenetic gene silencing. Extrinsic anti-silencing mechanisms involving histone demethylation or boundary elements help to limit the spread of inappropriate H3K9me12-15. However, how H3K9 methyltransferase activity is locally restricted or prevented from initiating random H3K9me-which would lead to aberrant gene silencing and epigenetic instability-is not fully understood. Here we reveal an autoinhibited conformation in the conserved H3K9 methyltransferase Clr4 (also known as Suv39h) of the fission yeast Schizosaccharomyces pombe that has a critical role in preventing aberrant heterochromatin formation. Biochemical and X-ray crystallographic data show that an internal loop in Clr4 inhibits the catalytic activity of this enzyme by blocking the histone H3K9 substrate-binding pocket, and that automethylation of specific lysines in this loop promotes a conformational switch that enhances the H3K9me activity of Clr4. Mutations that are predicted to disrupt this regulation lead to aberrant H3K9me, loss of heterochromatin domains and inhibition of growth, demonstrating the importance of the intrinsic inhibition and auto-activation of Clr4 in regulating the deposition of H3K9me and in preventing epigenetic instability. Conservation of the Clr4 autoregulatory loop in other H3K9 methyltransferases and the automethylation of a corresponding lysine in the human SUV39H2 homologue16 suggest that the mechanism described here is broadly conserved.
| S-EPMC6287498 | biostudies-literature
Project description:Remodelling of the peptidoglycan (PG) exoskeleton is intimately tied to the growth and division of bacteria. Enzymes that hydrolyse PG are critical for these processes, but their activities must be tightly regulated to prevent the generation of lethal breaches in the PG matrix. Despite their importance, the mechanisms regulating PG hydrolase activity have remained elusive. Here we investigate the control of cell division hydrolases called amidases (AmiA, AmiB and AmiC) required for Escherichia coli cell division. Poorly regulated amiB mutants were isolated encoding lytic AmiB variants with elevated basal PG hydrolase activities in vitro. The structure of an AmiB orthologue was also solved, revealing that the active site of AmiB is occluded by a conserved alpha helix. Strikingly, most of the amino acid substitutions in the lytic AmiB variants mapped to this domain and are predicted to disrupt its interaction with the active site. Our results therefore support a model in which cell separation is stimulated by the reversible relief of amidase autoinhibition governed by conserved subcomplexes within the cytokinetic ring. Analogous conformational control mechanisms are likely to be part of a general strategy used to control PG hydrolases present within multienzyme PG-remodelling machines.
| S-EPMC3418388 | biostudies-literature
Project description:During synaptic vesicle fusion, the soluble N-ethylmaleimide-sensitive factor-attachment protein receptor (SNARE) protein syntaxin-1 exhibits two conformations that both bind to Munc18-1: a "closed" conformation outside the SNARE complex and an "open" conformation in the SNARE complex. Although SNARE complexes containing open syntaxin-1 and Munc18-1 are essential for exocytosis, the function of closed syntaxin-1 is unknown. We generated knockin/knockout mice that expressed only open syntaxin-1B. Syntaxin-1B(Open) mice were viable but succumbed to generalized seizures at 2 to 3 months of age. Binding of Munc18-1 to syntaxin-1 was impaired in syntaxin-1B(Open) synapses, and the size of the readily releasable vesicle pool was decreased; however, the rate of synaptic vesicle fusion was dramatically enhanced. Thus, the closed conformation of syntaxin-1 gates the initiation of the synaptic vesicle fusion reaction, which is then mediated by SNARE-complex/Munc18-1 assemblies.
| S-EPMC3235364 | biostudies-literature
Project description:The Polycomb Repressive Complex 2 (PRC2) methylates H3K27 to regulate development and cell fate by transcriptional silencing. Alteration of PRC2 is associated with various cancers. Here, we show that mouse Kdm1a deletion causes a dramatic reduction of PRC2 proteins, whereas mouse null mutation of L3mbtl3 or Dcaf5 results in PRC2 accumulation and increased H3K27 trimethylation. The catalytic subunit of PRC2, EZH2, is methylated at lysine 20 (K20), promoting EZH2 proteolysis by L3MBTL3 and the CLR4DCAF5 ubiquitin ligase. KDM1A (LSD1) demethylates the methylated K20 to stabilize EZH2. K20 methylation is inhibited by AKT-mediated phosphorylation of serine 21 in EZH2. Mouse Ezh2K20R/K20R mutants develop hepatosplenomegaly associated with high GFI1B expression, and Ezh2K20R/K20R mutant bone marrows expand hematopoietic stem cells and downstream hematopoietic populations. Our studies reveal that EZH2 is regulated by methylation-dependent proteolysis, which is negatively controlled by AKT-mediated S21 phosphorylation to establish a methylation-phosphorylation switch to regulate the PRC2 activity and hematopoiesis.
| S-EPMC10901513 | biostudies-literature
Project description:Bacterial RNA polymerases (RNAPs) are targets for antibiotics. Myxopyronin binds to the RNAP switch regions to block structural rearrangements needed for formation of open promoter complexes. Bacterial RNAPs containing the major variant σ(54) factor are activated by enhancer-binding proteins (bEBPs) and transcribe genes whose products are needed in pathogenicity and stress responses. We show that (i) enhancer-dependent RNAPs help Escherichia coli to survive in the presence of myxopyronin, (ii) enhancer-dependent RNAPs partially resist inhibition by myxopyronin and (iii) ATP hydrolysis catalysed by bEBPs is obligatory for functional interaction of the RNAP switch regions with the transcription start site. We demonstrate that enhancer-dependent promoters contain two barriers to full DNA opening, allowing tight regulation of transcription initiation. bEBPs engage in a dual switch to (i) allow propagation of nucleated DNA melting from an upstream DNA fork junction and (ii) complete the formation of the transcription bubble and downstream DNA fork junction at the RNA synthesis start site, resulting in switch region-dependent RNAP clamp closure and open promoter complex formation.
| S-EPMC3505966 | biostudies-literature
Project description:Inspired by the rigidified architecture of 'picket-fence' systems, we propose a strategy utilizing strain to impose intramolecular tension in already peripherally overcrowded structures leading to selective atropisomeric conversion. Employing this approach, tuneable shape-persistent porphyrin conformations were acquired exhibiting distinctive supramolecular nanostructures based on the orientation of the peripheral groups. The intrinsic assemblies driven by non-covalent bonding interactions form supramolecular polymers while encapsulating small molecules in parallel channels or solvent-accessible voids. The developed molecular strain engineering methodologies combined with synthetic approaches have allowed the introduction of the pivalate units creating a highly strained molecular skeleton. Changes in the absorption spectrum indicated the presence of severe steric repulsions between the peripheral groups which were confirmed by single crystal X-ray analysis. To release the steric strain introduced by the peripheral units, thermal equilibration strategies were used to selectively convert the most abundant atropisomer to the desirable minor one.
| S-EPMC9299809 | biostudies-literature
Project description:The interaction of the signal-recognition particle (SRP) with its receptor (SR) mediates co-translational protein targeting to the membrane. SRP and SR interact via their homologous core GTPase domains and N-terminal four-helix bundles (N domains). SRP-SR complex formation is slow unless catalyzed by SRP's essential RNA component. We show that truncation of the first helix of the N domain (helix N1) of both proteins dramatically accelerates their interaction. SRP and SR with helix N1 truncations interact at nearly the RNA-catalyzed rate in the absence of RNA. NMR spectroscopy and analysis of GTPase activity show that helix N1 truncation in SR mimics the conformational switch caused by complex formation. These results demonstrate that the N-terminal helices of SRP and SR are autoinhibitory for complex formation in the absence of SRP RNA, suggesting a mechanism for RNA-mediated coordination of the SRP-SR interaction.
| S-EPMC2767265 | biostudies-literature
Project description:Cellular genomic DNA is replicated by a multiprotein replisome machine. The replisome contains numerous essential factors that unwind, prime and synthesize each of the two strands of duplex DNA. The antiparallel structure of DNA, and unidirectional activity of DNA polymerases, requires the two strands of DNA to be extended in opposite directions, and this structural feature requires distinctive processes for synthesis of the two strands. Genome duplication is of central importance to all cell types, and one may expect the replisome apparatus to be conserved from bacteria to human, as is the case with RNA polymerase driven transcription and ribosome mediated translation. However, it is known that the replication factors of bacteria are not homologous to those of archaea and eukaryotes, indicating that the replication process evolved twice, independently, rather than from a common ancestor cell. Thus, the different domains of life may exhibit significant differences in their mechanistic strategy of replication. In this review, we compare and contrast the different structures and mechanistic features of the cellular replication machinery in the three domains of life.
| S-EPMC5199024 | biostudies-literature
Project description:The flagellar motor can spin in both counterclockwise (CCW) and clockwise (CW) directions. The flagellar motor consists of a rotor and multiple stator units, which act as a proton channel. The rotor is composed of the transmembrane MS ring made of FliF and the cytoplasmic C ring consisting of FliG, FliM, and FliN. The C ring is directly involved in rotation and directional switching. The Salmonella FliF-FliG deletion fusion motor missing 56 residues from the C terminus of FliF and 94 residues from the N terminus of FliG keeps a domain responsible for the interaction with the stator intact, but its motor function is reduced significantly. Here, we report the structure and function of the FliF-FliG deletion fusion motor. The FliF-FliG deletion fusion not only resulted in a strong CW switch bias but also affected rotor-stator interactions coupled with proton translocation through the proton channel of the stator unit. The energy coupling efficiency of the deletion fusion motor was the same as that of the wild-type motor. Extragenic suppressor mutations in FliG, FliM, or FliN not only relieved the strong CW switch bias but also increased the motor speed at low load. The FliF-FliG deletion fusion made intersubunit interactions between C ring proteins tighter compared to the wild-type motor, whereas the suppressor mutations affect such tighter intersubunit interactions. We propose that a change of intersubunit interactions between the C ring proteins may be required for high-speed motor rotation as well as direction switching.IMPORTANCE The bacterial flagellar motor is a bidirectional rotary motor for motility and chemotaxis, which often plays an important role in infection. The motor is a large transmembrane protein complex composed of a rotor and multiple stator units, which also act as a proton channel. Motor torque is generated through their cyclic association and dissociation coupled with proton translocation through the proton channel. A large cytoplasmic ring of the motor, called C ring, is responsible for rotation and switching by interacting with the stator, but the mechanism remains unknown. By analyzing the structure and function of the wild-type motor and a mutant motor missing part of the C ring connecting itself with the transmembrane rotor ring while keeping a stator-interacting domain for bidirectional torque generation intact, we found interesting clues to the change in the C ring conformation for the switching and rotation involving loose and tight intersubunit interactions.
| S-EPMC6445934 | biostudies-literature