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1.
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2.
  • Böiers, Charlotta, et al. (author)
  • Lymphomyeloid Contribution of an Immune-Restricted Progenitor Emerging Prior to Definitive Hematopoietic Stem Cells.
  • 2013
  • In: Cell Stem Cell. - : Elsevier BV. - 1934-5909 .- 1875-9777. ; 13:5, s. 535-548
  • Journal article (peer-reviewed)abstract
    • In jawed vertebrates, development of an adaptive immune-system is essential for protection of the born organism against otherwise life-threatening pathogens. Myeloid cells of the innate immune system are formed early in development, whereas lymphopoiesis has been suggested to initiate much later, following emergence of definitive hematopoietic stem cells (HSCs). Herein, we demonstrate that the embryonic lymphoid commitment process initiates earlier than previously appreciated, prior to emergence of definitive HSCs, through establishment of a previously unrecognized entirely immune-restricted and lymphoid-primed progenitor. Notably, this immune-restricted progenitor appears to first emerge in the yolk sac and contributes physiologically to the establishment of lymphoid and some myeloid components of the immune-system, establishing the lymphomyeloid lineage restriction process as an early and physiologically important lineage-commitment step in mammalian hematopoiesis.
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3.
  • Aleksandrova, Elena V, et al. (author)
  • Structural basis of Cfr-mediated antimicrobial resistance and mechanisms for its evasion
  • 2023
  • Other publication (other academic/artistic)abstract
    • The ribosome is an essential drug target as many classes of clinically important antibiotics bind and inhibit its functional centers. The catalytic peptidyl transferase center (PTC) is targeted by the broadest array of inhibitors belonging to several chemical classes. One of the most abundant and clinically prevalent mechanisms of resistance to PTC-acting drugs is C8-methylation of the universally conserved adenine residue 2503 (A2503) of the 23S rRNA by the methyltransferase Cfr. Despite its clinical significance, a sufficient understanding of the molecular mechanisms underlying Cfr-mediated resistance is currently lacking. In this work, we developed a method to express a functionally-active Cfr-methyltransferase in the thermophilic bacterium Thermus thermophilus and report a set of high-resolution structures of the Cfr-modified 70S ribosome containing aminoacyl- and peptidyl-tRNAs. Our structures reveal that an allosteric rearrangement of nucleotide A2062 upon Cfr-methylation of A2503 is likely responsible for the inability of some PTC inhibitors to bind to the ribosome, providing additional insights into the Cfr resistance mechanism. Lastly, by determining the structures of the Cfr-methylated ribosome in complex with the antibiotics iboxamycin and tylosin, we provide the structural bases behind two distinct mechanisms of evading Cfr-mediated resistance.
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4.
  • Aleksandrova, Elena V., et al. (author)
  • Structural basis of Cfr-mediated antimicrobial resistance and mechanisms to evade it
  • 2024
  • In: Nature Chemical Biology. - : Springer Nature. - 1552-4450 .- 1552-4469. ; 20:7, s. 867-876
  • Journal article (peer-reviewed)abstract
    • The bacterial ribosome is an essential drug target as many clinically important antibiotics bind and inhibit its functional centers. The catalytic peptidyl transferase center (PTC) is targeted by the broadest array of inhibitors belonging to several chemical classes. One of the most abundant and clinically prevalent resistance mechanisms to PTC-acting drugs in Gram-positive bacteria is C8-methylation of the universally conserved A2503 nucleobase by Cfr methylase in 23S ribosomal RNA. Despite its clinical importance, a sufficient understanding of the molecular mechanisms underlying Cfr-mediated resistance is currently lacking. Here, we report a set of high-resolution structures of the Cfr-modified 70S ribosome containing aminoacyl- and peptidyl-transfer RNAs. These structures reveal an allosteric rearrangement of nucleotide A2062 upon Cfr-mediated methylation of A2503 that likely contributes to the reduced potency of some PTC inhibitors. Additionally, we provide the structural bases behind two distinct mechanisms of engaging the Cfr-methylated ribosome by the antibiotics iboxamycin and tylosin. 
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5.
  • Obana, Nozomu, et al. (author)
  • Genome-encoded ABCF factors implicated in intrinsic antibiotic resistance in Gram-positive bacteria : VmlR2, Ard1 and CplR
  • 2023
  • In: Nucleic Acids Research. - : Oxford University Press (OUP). - 1362-4962 .- 0305-1048. ; 51:9, s. 4536-4554
  • Journal article (peer-reviewed)abstract
    • Genome-encoded antibiotic resistance (ARE) ATP-binding cassette (ABC) proteins of the F subfamily (ARE-ABCFs) mediate intrinsic resistance in diverse Gram-positive bacteria. The diversity of chromosomally-encoded ARE-ABCFs is far from being fully experimentally explored. Here we characterise phylogenetically diverse genome-encoded ABCFs from Actinomycetia (Ard1 from Streptomyces capreolus, producer of the nucleoside antibiotic A201A), Bacilli (VmlR2 from soil bacterium Neobacillus vireti) and Clostridia (CplR from Clostridium perfringens, Clostridium sporogenes and Clostridioides difficile). We demonstrate that Ard1 is a narrow spectrum ARE-ABCF that specifically mediates self-resistance against nucleoside antibiotics. The single-particle cryo-EM structure of a VmlR2-ribosome complex allows us to rationalise the resistance spectrum of this ARE-ABCF that is equipped with an unusually long antibiotic resistance determinant (ARD) subdomain. We show that CplR contributes to intrinsic pleuromutilin, lincosamide and streptogramin A resistance in Clostridioides, and demonstrate that C. difficile CplR (CDIF630_02847) synergises with the transposon-encoded 23S ribosomal RNA methyltransferase Erm to grant high levels of antibiotic resistance to the C. difficile 630 clinical isolate. Finally, assisted by uORF4u, our novel tool for detection of upstream open reading frames, we dissect the translational attenuation mechanism that controls the induction of cplR expression upon an antibiotic challenge.
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6.
  • Ainelo, Andres, et al. (author)
  • The structure of DarB in complex with RelNTD reveals nonribosomal activation of Rel stringent factors
  • 2023
  • In: Science Advances. - : American Association for the Advancement of Science (AAAS). - 2375-2548. ; 9:3, s. 1-14
  • Journal article (peer-reviewed)abstract
    • Rel stringent factors are bifunctional ribosome-associated enzymes that catalyze both synthesis and hydrolysis of the alarmones (p)ppGpp. Besides the allosteric control by starved ribosomes and (p)ppGpp, Rel is regulated by various protein factors depending on specific stress conditions, including the c-di-AMP-binding protein DarB. However, how these effector proteins control Rel remains unknown. We have determined the crystal structure of the DarB2:RelNTD2 complex, uncovering that DarB directly engages the SYNTH domain of Rel to stimulate (p)ppGpp synthesis. This association with DarB promotes a SYNTH-primed conformation of the N-terminal domain region, markedly increasing the affinity of Rel for ATP while switching off the hydrolase activity of the enzyme. Binding to c-di-AMP rigidifies DarB, imposing an entropic penalty that precludes DarB-mediated control of Rel during normal growth. Our experiments provide the basis for understanding a previously unknown mechanism of allosteric regulation of Rel stringent factors independent of amino acid starvation.
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7.
  • Ajawatanawong, Pravech, et al. (author)
  • SeqFIRE : a web application for automated extraction of indel regions and conserved blocks from protein multiple sequence alignments
  • 2012
  • In: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 40:W1, s. W340-W347
  • Journal article (peer-reviewed)abstract
    • Analyses of multiple sequence alignments generally focus on well-defined conserved sequence blocks, while the rest of the alignment is largely ignored or discarded. This is especially true in phylogenomics, where large multigene datasets are produced through automated pipelines. However, some of the most powerful phylogenetic markers have been found in the variable length regions of multiple alignments, particularly insertions/deletions (indels) in protein sequences. We have developed Sequence Feature and Indel Region Extractor (SeqFIRE) to enable the automated identification and extraction of indels from protein sequence alignments. The program can also extract conserved blocks and identify fast evolving sites using a combination of conservation and entropy. All major variables can be adjusted by the user, allowing them to identify the sets of variables most suited to a particular analysis or dataset. Thus, all major tasks in preparing an alignment for further analysis are combined in a single flexible and user-friendly program. The output includes a numbered list of indels, alignments in NEXUS format with indels annotated or removed and indel-only matrices. SeqFIRE is a user-friendly web application, freely available online at www.seqfire.org/.
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8.
  • Atkinson, Gemma C., et al. (author)
  • An evolutionary ratchet leading to loss of elongation factors in eukaryotes
  • 2014
  • In: BMC Evolutionary Biology. - : Springer Science and Business Media LLC. - 1471-2148. ; 14, s. 1-9
  • Journal article (peer-reviewed)abstract
    • Background: The GTPase eEF1A is the eukaryotic factor responsible for the essential, universal function of aminoacyl-tRNA delivery to the ribosome. Surprisingly, eEF1A is not universally present in eukaryotes, being replaced by the paralog EFL independently in multiple lineages. The driving force behind this unusually frequent replacement is poorly understood. Results: Through sequence searching of genomic and EST databases, we find a striking association of eEF1A replacement by EFL and loss of eEF1A's guanine exchange factor, eEF1Bα, suggesting that EFL is able to spontaneously recharge with GTP. Sequence conservation and homology modeling analyses indicate several sequence regions that may be responsible for EFL's lack of requirement for eEF1Bα. Conclusions: We propose that the unusual pattern of eEF1A, eEF1Bα and EFL presence and absence can be explained by a ratchet-like process: if either eEF1A or eEF1Bα diverges beyond functionality in the presence of EFL, the system is unable to return to the ancestral, eEF1A:eEFBα-driven state.
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9.
  • Atkinson, Gemma C., et al. (author)
  • Evolution of nonstop, no-go and nonsense-mediated mRNA decay and their termination factor-derived components
  • 2008
  • In: BMC Evolutionary Biology. - : Springer Science and Business Media LLC. - 1471-2148. ; 8, s. 290-
  • Journal article (peer-reviewed)abstract
    • Background: Members of the eukaryote/archaea specific eRF1 and eRF3 protein families have central roles in translation termination. They are also central to various mRNA surveillance mechanisms, together with the eRF1 paralogue Dom34p and the eRF3 paralogues Hbs1p and Ski7p. We have examined the evolution of eRF1 and eRF3 families using sequence similarity searching, multiple sequence alignment and phylogenetic analysis. Results: Extensive BLAST searches confirm that Hbs1p and eRF3 are limited to eukaryotes, while Dom34p and eRF1 (a/eRF1) are universal in eukaryotes and archaea. Ski7p appears to be restricted to a subset of Saccharomyces species. Alignments show that Dom34p does not possess the characteristic class-1 RF minidomains GGQ, NIKS and YXCXXXF, in line with recent crystallographic analysis of Dom34p. Phylogenetic trees of the protein families allow us to reconstruct the evolution of mRNA surveillance mechanisms mediated by these proteins in eukaryotes and archaea. Conclusion: We propose that the last common ancestor of eukaryotes and archaea possessed Dom34p-mediated no-go decay (NGD). This ancestral Dom34p may or may not have required a trGTPase, mostly like a/eEF1A, for its delivery to the ribosome. At an early stage in eukaryotic evolution, eEF1A was duplicated, giving rise to eRF3, which was recruited for translation termination, interacting with eRF1. eRF3 evolved nonsense-mediated decay (NMD) activity either before or after it was again duplicated, giving rise to Hbs1p, which we propose was recruited to assist eDom34p in eukaryotic NGD. Finally, a third duplication within ascomycete yeast gave rise to Ski7p, which may have become specialised for a subset of existing Hbs1p functions in non-stop decay (NSD). We suggest Ski7p-mediated NSD may be a specialised mechanism for counteracting the effects of increased stop codon read-through caused by prion-domain [ PSI+] mediated eRF3 precipitation.
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10.
  • Beljantseva, Jelena, et al. (author)
  • Negative allosteric regulation of Enterococcus faecalis small alarmone synthetase RelQ by single-stranded RNA
  • 2017
  • In: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 114:14, s. 3726-3731
  • Journal article (peer-reviewed)abstract
    • The alarmone nucleotides guanosine pentaphosphate (pppGpp) and tetraphosphate (ppGpp), collectively referred to as (p)ppGpp, are key regulators of bacterial growth, stress adaptation, pathogenicity, and antibiotic tolerance. We show that the tetrameric small alarmone synthetase (SAS) RelQ from the Gram-positive pathogen Enterococcus faecalis is a sequence-specific RNA-binding protein. RelQ's enzymatic and RNA binding activities are subject to intricate allosteric regulation. (p)ppGpp synthesis is potently inhibited by the binding of single-stranded RNA. Conversely, RelQ's enzymatic activity destabilizes the RelQ: RNA complex. pppGpp, an allosteric activator of the enzyme, counteracts the effect of RNA. Tetramerization of RelQ is essential for this regulatory mechanism, because both RNA binding and enzymatic activity are abolished by deletion of the SAS-specific C-terminal helix 5 alpha. The interplay of pppGpp binding, (p)ppGpp synthesis, and RNA binding unites two archetypal regulatory paradigms within a single protein. The mechanism is likely a prevalent but previously unappreciated regulatory switch used by the widely distributed bacterial SAS enzymes.
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11.
  • Crowe-McAuliffe, Caillan, et al. (author)
  • Structural basis for antibiotic resistance mediated by the Bacillus subtilis ABCF ATPase VmlR
  • 2018
  • In: Proceedings of the National Academy of Sciences of the United States of America. - : National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 115:36, s. 8978-8983
  • Journal article (peer-reviewed)abstract
    • Many Gram-positive pathogenic bacteria employ ribosomal protection proteins (RPPs) to confer resistance to clinically important antibiotics. In Bacillus subtilis, the RPP VmlR confers resistance to lincomycin (Lnc) and the streptogramin A (SA) antibiotic virginiamycin M (VgM). VmlR is an ATP-binding cassette (ABC) protein of the F type, which, like other antibiotic resistance (ARE) ABCF proteins, is thought to bind to antibiotic-stalled ribosomes and promote dissociation of the drug from its binding site. To investigate the molecular mechanism by which VmlR confers antibiotic resistance, we have determined a cryo-electron microscopy (cryo-EM) structure of an ATPase-deficient B. subtilis VmlR-EQ(2) mutant in complex with a B. subtilis ErmDL-stalled ribosomal complex (SRC). The structure reveals that VmlR binds within the E site of the ribosome, with the antibiotic resistance domain (ARD) reaching into the peptidyltransferase center (PTC) of the ribosome and a C-terminal extension (CTE) making contact with the small subunit (SSU). To access the PTC, VmlR induces a conformational change in the P-site tRNA, shifting the acceptor arm out of the PTC and relocating the CCA end of the P-site tRNA toward the A site. Together with microbiological analyses, our study indicates that VmlR allosterically dissociates the drug from its ribosomal binding site and exhibits specificity to dislodge VgM, Lnc, and the pleuromutilin tiamulin (Tia), but not chloramphenicol (Cam), linezolid (Lnz), nor the macrolide erythromycin (Ery).
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12.
  • Crowe-McAuliffe, Caillan, et al. (author)
  • Structural Basis for Bacterial Ribosome-Associated Quality Control by RqcH and RqcP
  • 2021
  • In: Molecular Cell. - : Cell Press. - 1097-2765 .- 1097-4164. ; 81:1, s. 115-126.e7
  • Journal article (peer-reviewed)abstract
    • In all branches of life, stalled translation intermediates are recognized and processed by ribosome-associated quality control (RQC) pathways. RQC begins with the splitting of stalled ribosomes, leaving an unfinished polypeptide still attached to the large subunit. Ancient and conserved NEMF family RQC proteins target these incomplete proteins for degradation by the addition of C-terminal "tails.'' How such tailing can occur without the regular suite of translational components is, however, unclear. Using single-particle cryo-electron microscopy (EM) of native complexes, we show that C-terminal tailing in Bacillus subtilis is mediated by NEMF protein RqcH in concert with RqcP, an Hsp15 family protein. Our structures reveal how these factors mediate tRNA movement across the ribosomal 50S subunit to synthesize polypeptides in the absence of mRNA or the small subunit.
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13.
  • Crowe-McAuliffe, Caillan, et al. (author)
  • Structural basis for PoxtA-mediated resistance to phenicol and oxazolidinone antibiotics
  • 2022
  • In: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 13
  • Journal article (peer-reviewed)abstract
    • PoxtA and OptrA are ATP binding cassette (ABC) proteins of the F subtype (ABCF). They confer resistance to oxazolidinone and phenicol antibiotics, such as linezolid and chloramphenicol, which stall translating ribosomes when certain amino acids are present at a defined position in the nascent polypeptide chain. These proteins are often encoded on mobile genetic elements, facilitating their rapid spread amongst Gram-positive bacteria, and are thought to confer resistance by binding to the ribosome and dislodging the bound antibiotic. However, the mechanistic basis of this resistance remains unclear. Here we refine the PoxtA spectrum of action, demonstrate alleviation of linezolid-induced context-dependent translational stalling, and present cryo-electron microscopy structures of PoxtA in complex with the Enterococcus faecalis 70S ribosome. PoxtA perturbs the CCA-end of the P-site tRNA, causing it to shift by ∼4 Å out of the ribosome, corresponding to a register shift of approximately one amino acid for an attached nascent polypeptide chain. We postulate that the perturbation of the P-site tRNA by PoxtA thereby alters the conformation of the attached nascent chain to disrupt the drug binding site.
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14.
  • Crowe-McAuliffe, Caillan, et al. (author)
  • Structural basis of ABCF-mediated resistance to pleuromutilin, lincosamide, and streptogramin A antibiotics in Gram-positive pathogens
  • 2021
  • In: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 12:1
  • Journal article (peer-reviewed)abstract
    • Target protection proteins confer resistance to the host organism by directly binding to the antibiotic target. One class of such proteins are the antibiotic resistance (ARE) ATP-binding cassette (ABC) proteins of the F-subtype (ARE-ABCFs), which are widely distributed throughout Gram-positive bacteria and bind the ribosome to alleviate translational inhibition from antibiotics that target the large ribosomal subunit. Here, we present single-particle cryo-EM structures of ARE-ABCF-ribosome complexes from three Gram-positive pathogens: Enterococcus faecalis LsaA, Staphylococcus haemolyticus VgaALC and Listeria monocytogenes VgaL. Supported by extensive mutagenesis analysis, these structures enable a general model for antibiotic resistance mediated by these ARE-ABCFs to be proposed. In this model, ABCF binding to the antibiotic-stalled ribosome mediates antibiotic release via mechanistically diverse long-range conformational relays that converge on a few conserved ribosomal RNA nucleotides located at the peptidyltransferase center. These insights are important for the future development of antibiotics that overcome such target protection resistance mechanisms.
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15.
  • Dominguez-Molina, Lucia, et al. (author)
  • Mechanisms of neutralization of toxSAS from toxin-antitoxin modules
  • In: Nature Chemical Biology. - 1552-4469.
  • Journal article (peer-reviewed)abstract
    • Toxic small alarmone synthetase (toxSAS) enzymes constitute a family of bacterial effectors present in toxin-antitoxin and secretion systems. toxSASs act through either translation inhibition mediated by pyrophosphorylation of transfer RNA (tRNA) CCA ends or synthesis of the toxic alarmone adenosine pentaphosphate ((pp)pApp) and adenosine triphosphate (ATP) depletion, exemplified by FaRel2 and FaRel, respectively. However, structural bases of toxSAS neutralization are missing. Here we show that the pseudo-Zn 2+ finger domain (pZFD) of the ATfaRel2 antitoxin precludes access of ATP to the pyrophosphate donor site of the FaRel2 toxin, without affecting recruitment of the tRNA pyrophosphate acceptor. By contrast, (pp)pApp-producing toxSASs are inhibited by Tis1 antitoxin domains though occlusion of the pyrophosphate acceptor-binding site. Consequently, the auxiliary pZFD of AT2faRel is dispensable for FaRel neutralization. Collectively, our study establishes the general principles of toxSAS inhibition by structured antitoxin domains, with the control strategy directly coupled to toxSAS substrate specificity.
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16.
  • Egorov, Artyom A., et al. (author)
  • uORF4u : a tool for annotation of conserved upstream open reading frames
  • 2023
  • In: Bioinformatics. - 1367-4803. ; 39:5
  • Journal article (peer-reviewed)abstract
    • Summary: Upstream open reading frames (uORFs, often encoding so-called leader peptides) can regulate translation and transcription of downstream main ORFs (mORFs) in prokaryotes and eukaryotes. However, annotation of novel functional uORFs is challenging due to their short size of usually <100 codons. While transcription- and translation-level next-generation sequencing methods can be used for genome-wide functional uORF identification, this data are not available for the vast majority of species with sequenced genomes. At the same time, the exponentially increasing amount of genome assemblies gives us the opportunity to take advantage of evolutionary conservation in our predictions of functional ORFs. Here, we present a tool for conserved uORF annotation in 50 upstream sequences of a user-defined protein of interest or a set of protein homologs. It can also be used to find small conserved ORFs within a set of nucleotide sequences. The output includes publication-quality figures with multiple sequence alignments, sequence logos, and locus annotation of the predicted conserved uORFs in graphical vector format. Availability and implementation: uORF4u is written in Python3 and runs on Linux and MacOS. The command-line interface covers most practical use cases, while the provided Python API allows usage within a Python program and additional customization. Source code is available from the GitHub page: github.com/GCA-VH-lab/uorf4u. Detailed documentation that includes an example-driven guide available at the software home page: gca-vh-lab.github.io/uorf4u. A web version of uORF4u is available at server.atkinson-lab.com/uorf4u.
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17.
  • Ernits, Karin, et al. (author)
  • The structural basis of hyperpromiscuity in a core combinatorial network of type II toxin-antitoxin and related phage defense systems
  • 2023
  • In: Proceedings of the National Academy of Sciences of the United States of America. - 1091-6490 .- 0027-8424. ; 120:33, s. 1-12
  • Journal article (peer-reviewed)abstract
    • Toxin-antitoxin (TA) systems are a large group of small genetic modules found in prokaryotes and their mobile genetic elements. Type II TAs are encoded as bicistronic (two-gene) operons that encode two proteins: a toxin and a neutralizing antitoxin. Using our tool NetFlax (standing for Network-FlaGs for toxins and antitoxins), we have performed a large-scale bioinformatic analysis of proteinaceous TAs, revealing interconnected clusters constituting a core network of TA-like gene pairs. To understand the structural basis of toxin neutralization by antitoxins, we have predicted the structures of 3,419 complexes with AlphaFold2. Together with mutagenesis and functional assays, our structural predictions provide insights into the neutralizing mechanism of the hyperpromiscuous Panacea antitoxin domain. In antitoxins composed of standalone Panacea, the domain mediates direct toxin neutralization, while in multidomain antitoxins the neutralization is mediated by other domains, such as PAD1, Phd-C, and ZFD. We hypothesize that Panacea acts as a sensor that regulates TA activation. We have experimentally validated 16 NetFlax TA systems and used domain annotations and metabolic labeling assays to predict their potential mechanisms of toxicity (such as membrane disruption, and inhibition of cell division or protein synthesis) as well as biological functions (such as antiphage defense). We have validated the antiphage activity of a RosmerTA system encoded by Gordonia phage Kita, and used fluorescence microscopy to confirm its predicted membrane-depolarizing activity. The interactive version of the NetFlax TA network that includes structural predictions can be accessed at http://netflax.webflags.se/.
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18.
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19.
  • Hauryliuk, Vasili, 1980-, et al. (author)
  • Recent functional insights into the role of (p)ppGpp in bacterial physiology
  • 2015
  • In: Nature Reviews Microbiology. - : Macmillan Publishers Ltd.. - 1740-1526 .- 1740-1534. ; 13:5, s. 298-309
  • Research review (peer-reviewed)abstract
    • The alarmones guanosine tetraphosphate and guanosine pentaphosphate (collectively referred to as (p) ppGpp) are involved in regulating growth and several different stress responses in bacteria. In recent years, substantial progress has been made in our understanding of the molecular mechanisms of (p) ppGpp metabolism and (p) ppGpp-mediated regulation. In this Review, we summarize these recent insights, with a focus on the molecular mechanisms governing the activity of the RelA/SpoT homologue (RSH) proteins, which are key players that regulate the cellular levels of (p) ppGpp. We also discuss the structural basis of transcriptional regulation by (p) ppGpp and the role of (p) ppGpp in GTP metabolism and in the emergence of bacterial persisters.
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20.
  • Hauryliuk, Vasili, et al. (author)
  • Small Alarmone Synthetases as novel bacterial RNA-binding proteins
  • 2017
  • In: RNA Biology. - : Taylor & Francis. - 1547-6286 .- 1555-8584. ; 14:12, s. 1695-1699
  • Research review (peer-reviewed)abstract
    • The alarmone nucleotides guanosine pentaphosphate (pppGpp) and tetraphosphate (ppGpp), collectively referred to as (p)ppGpp, are key regulators of bacterial growth, stress adaptation, antibiotic tolerance and pathogenicity. We have recently shown that the Small Alarmone Synthetase (SAS) RelQ from the Gram-positive pathogen Enterococcus faecalis has an RNA-binding activity (Beljantseva et al. 2017). RelQ's activities as an enzyme and as an RNA-binding protein are mutually incompatible: binding of single-stranded RNA potently inhibits (p)ppGpp synthesis in a sequence-specific manner, and RelQ's enzymatic activity destabilizes the RNA: RelQ complex. RelQ's allosteric regulator, pppGpp, destabilizes RNA binding and activates RelQ's enzymatic activity. Since SAS enzymes are widely distributed in bacteria, and, as has been discovered recently, are also mobilized by phages (Dedrick et al. 2017), RNA binding to SASs could be a widespread mechanism. The initial discovery raises numerous questions regarding RNA-binding function of the SAS enzymes: What is the molecular mechanism underlying the incompatibility of RNA: SAS complex formation with pppGpp binding and (p)ppGpp synthesis? What are the RNA targets in living cells? What is the regulatory output of the system - (p)ppGpp synthesis, modulation of RNA structure and function, or both?
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21.
  • Jimmy, Steffi, et al. (author)
  • A widespread toxin-antitoxin system exploiting growth control via alarmone signaling
  • 2020
  • In: Proceedings of the National Academy of Sciences of the United States of America. - : National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 117:19, s. 10500-10510
  • Journal article (peer-reviewed)abstract
    • Under stressful conditions, bacterial RelA-SpoT Homolog (RSH) enzymes synthesize the alarmone (p)ppGpp, a nucleotide second messenger. (p)ppGpp rewires bacterial transcription and metabolism to cope with stress, and, at high concentrations, inhibits the process of protein synthesis and bacterial growth to save and redirect resources until conditions improve. Single-domain small alarmone synthetases (SASs) are RSH family members that contain the (p)ppGpp synthesis (SYNTH) domain, but lack the hydrolysis (HD) domain and regulatory C-terminal domains of the long RSHs such as Rel, RelA, and SpoT. We asked whether analysis of the genomic context of SASs can indicate possible functional roles. Indeed, multiple SAS subfamilies are encoded in widespread conserved bicistronic operon architectures that are reminiscent of those typically seen in toxin-antitoxin (TA) operons. We have validated five of these SASs as being toxic (toxSASs), with neutralization by the protein products of six neighboring antitoxin genes. The toxicity of Cellulomonas marina toxSAS FaRel is mediated by the accumulation of alarmones ppGpp and ppApp, and an associated depletion of cellular guanosine triphosphate and adenosine triphosphate pools, and is counteracted by its HD domain-containing antitoxin. Thus, the ToxSAS-antiToxSAS system with its multiple different antitoxins exemplifies how ancient nucleotide-based signaling mechanisms can be repurposed as TA modules during evolution, potentially multiple times independently.
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22.
  • Kasari, Villu, et al. (author)
  • A role for the Saccharomyces cerevisiae ABCF protein New1 in translation termination/recycling
  • 2019
  • In: Nucleic Acids Research. - : Oxford University Press. - 0305-1048 .- 1362-4962. ; 47:16, s. 8807-8820
  • Journal article (peer-reviewed)abstract
    • Translation is controlled by numerous accessory proteins and translation factors. In the yeast Saccharomyces cerevisiae, translation elongation requires an essential elongation factor, the ABCF ATPase eEF3. A closely related protein, New1, is encoded by a non-essential gene with cold sensitivity and ribosome assembly defect knock-out phenotypes. Since the exact molecular function of New1 is unknown, it is unclear if the ribosome assembly defect is direct, i.e. New1 is a bona fide assembly factor, or indirect, for instance due to a defect in protein synthesis. To investigate this, we employed yeast genetics, cryo-electron microscopy (cryo-EM) and ribosome profiling (Ribo-Seq) to interrogate the molecular function of New1. Overexpression of New1 rescues the inviability of a yeast strain lacking the otherwise strictly essential translation factor eEF3. The structure of the ATPase-deficient (EQ2) New1 mutant locked on the 80S ribosome reveals that New1 binds analogously to the ribosome as eEF3. Finally, Ribo-Seq analysis revealed that loss of New1 leads to ribosome queuing upstream of 3′-terminal lysine and arginine codons, including those genes encoding proteins of the cytoplasmic translational machinery. Our results suggest that New1 is a translation factor that fine-tunes the efficiency of translation termination or ribosome recycling.
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23.
  • Kasari, Villu, et al. (author)
  • Ribosome profiling analysis of eEF3-depleted Saccharomyces cerevisiae
  • 2019
  • In: Scientific Reports. - : Nature Publishing Group. - 2045-2322. ; 9
  • Journal article (peer-reviewed)abstract
    • In addition to the standard set of translation factors common in eukaryotic organisms, protein synthesis in the yeast Saccharomyces cerevisiae requires an ABCF ATPase factor eEF3, eukaryotic Elongation Factor 3. eEF3 is an E-site binder that was originally identified as an essential factor involved in the elongation stage of protein synthesis. Recent biochemical experiments suggest an additional function of eEF3 in ribosome recycling. We have characterised the global effects of eEF3 depletion on translation using ribosome profiling. Depletion of eEF3 results in decreased ribosome density at the stop codon, indicating that ribosome recycling does not become rate limiting when eEF3 levels are low. Consistent with a defect in translation elongation, eEF3 depletion causes a moderate redistribution of ribosomes towards the 5' part of the open reading frames. We observed no E-site codon-or amino acid-specific ribosome stalling upon eEF3 depletion, supporting its role as a general elongation factor. Surprisingly, depletion of eEF3 leads to a relative decrease in P-site proline stalling, which we hypothesise is a secondary effect of generally decreased translation and/or decreased competition for the E-site with eIF5A.
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24.
  • Koller, Timm O, et al. (author)
  • Structural basis for HflXr-mediated antibiotic resistance in Listeria monocytogenes
  • 2022
  • In: Nucleic Acids Research. - : Oxford University Press (OUP). - 1362-4962 .- 0305-1048. ; 50:19, s. 11285-11300
  • Journal article (peer-reviewed)abstract
    • HflX is a ubiquitous bacterial GTPase that splits and recycles stressed ribosomes. In addition to HflX, Listeria monocytogenes contains a second HflX homolog, HflXr. Unlike HflX, HflXr confers resistance to macrolide and lincosamide antibiotics by an experimentally unexplored mechanism. Here, we have determined cryo-EM structures of L. monocytogenes HflXr-50S and HflX-50S complexes as well as L. monocytogenes 70S ribosomes in the presence and absence of the lincosamide lincomycin. While the overall geometry of HflXr on the 50S subunit is similar to that of HflX, a loop within the N-terminal domain of HflXr, which is two amino acids longer than in HflX, reaches deeper into the peptidyltransferase center. Moreover, unlike HflX, the binding of HflXr induces conformational changes within adjacent rRNA nucleotides that would be incompatible with drug binding. These findings suggest that HflXr confers resistance using an allosteric ribosome protection mechanism, rather than by simply splitting and recycling antibiotic-stalled ribosomes.
  •  
25.
  • Kononenko, Artem V., et al. (author)
  • GTP-dependent structural rearrangement of the eRF1:eRF3 complex and eRF3 sequence motifs essential for PABP binding
  • 2010
  • In: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 38:2, s. 548-558
  • Journal article (peer-reviewed)abstract
    • Translation termination in eukaryotes is governed by the concerted action of eRF1 and eRF3 factors. eRF1 recognizes the stop codon in the A site of the ribosome and promotes nascent peptide chain release, and the GTPase eRF3 facilitates this peptide release via its interaction with eRF1. In addition to its role in termination, eRF3 is involved in normal and nonsense-mediated mRNA decay through its association with cytoplasmic poly(A)-binding protein (PABP) via PAM2-1 and PAM2-2 motifs in the N-terminal domain of eRF3. We have studied complex formation between full-length eRF3 and its ligands (GDP, GTP, eRF1 and PABP) using isothermal titration calorimetry, demonstrating formation of the eRF1:eRF3:PABP:GTP complex. Analysis of the temperature dependence of eRF3 interactions with G nucleotides reveals major structural rearrangements accompanying formation of the eRF1:eRF3:GTP complex. This is in contrast to eRF1:eRF3:GDP complex formation, where no such rearrangements were detected. Thus, our results agree with the established active role of GTP in promoting translation termination. Through point mutagenesis of PAM2-1 and PAM2-2 motifs in eRF3, we demonstrate that PAM2-2, but not PAM2-1 is indispensible for eRF3:PABP complex formation.
  •  
26.
  • Kurata, Tatsuaki, et al. (author)
  • A hyperpromiscuous antitoxin protein domain for the neutralization of diverse toxin domains
  • 2022
  • In: Proceedings of the National Academy of Sciences of the United States of America. - : National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 119:6
  • Journal article (peer-reviewed)abstract
    • Toxin–antitoxin (TA) gene pairs are ubiquitous in microbial chromosomal genomes and plasmids as well as temperate bacteriophages. They act as regulatory switches, with the toxin limiting the growth of bacteria and archaea by compromising diverse essential cellular targets and the antitoxin counteracting the toxic effect. To uncover previously uncharted TA diversity across microbes and bacteriophages, we analyzed the conservation of genomic neighborhoods using our computational tool FlaGs (for flanking genes), which allows high-throughput detection of TA-like operons. Focusing on the widespread but poorly experimentally characterized antitoxin domain DUF4065, our in silico analyses indicated that DUF4065-containing proteins serve as broadly distributed antitoxin components in putative TA-like operons with dozens of different toxic domains with multiple different folds. Given the versatility of DUF4065, we have named the domain Panacea (and proteins containing the domain, PanA) after the Greek goddess of universal remedy. We have experimentally validated nine PanA-neutralized TA pairs. While the majority of validated PanA-neutralized toxins act as translation inhibitors or membrane disruptors, a putative nucleotide cyclase toxin from a Burkholderia prophage compromises transcription and translation as well as inducing RelA-dependent accumulation of the nucleotide alarmone (p)ppGpp. We find that Panacea-containing antitoxins form a complex with their diverse cognate toxins, characteristic of the direct neutralization mechanisms employed by Type II TA systems. Finally, through directed evolution, we have selected PanA variants that can neutralize noncognate TA toxins, thus experimentally demonstrating the evolutionary plasticity of this hyperpromiscuous antitoxin domain.
  •  
27.
  • Kuzmenko, Anton, et al. (author)
  • Aim-less translation : loss of Saccharomyces cerevisiae mitochondrial translation initiation factor mIF3/Aim23 leads to unbalanced protein synthesis
  • 2016
  • In: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 6
  • Journal article (peer-reviewed)abstract
    • The mitochondrial genome almost exclusively encodes a handful of transmembrane constituents of the oxidative phosphorylation (OXPHOS) system. Coordinated expression of these genes ensures the correct stoichiometry of the system's components. Translation initiation in mitochondria is assisted by two general initiation factors mIF2 and mIF3, orthologues of which in bacteria are indispensible for protein synthesis and viability. mIF3 was thought to be absent in Saccharomyces cerevisiae until we recently identified mitochondrial protein Aim23 as the missing orthologue. Here we show that, surprisingly, loss of mIF3/Aim23 in S. cerevisiae does not indiscriminately abrogate mitochondrial translation but rather causes an imbalance in protein production: the rate of synthesis of the Atp9 subunit of F1F0 ATP synthase (complex V) is increased, while expression of Cox1, Cox2 and Cox3 subunits of cytochrome c oxidase (complex IV) is repressed. Our results provide one more example of deviation of mitochondrial translation from its bacterial origins.
  •  
28.
  • Kuzmenko, Anton, et al. (author)
  • Mitochondrial translation initiation machinery : conservation and diversification
  • 2014
  • In: Biochimie. - : Elsevier. - 0300-9084 .- 1638-6183. ; 100C, s. 132-140
  • Journal article (peer-reviewed)abstract
    • The highly streamlined mitochondrial genome encodes almost exclusively a handful of transmembrane components of the respiratory chain complex. In order to ensure the correct assembly of the respiratory chain, the products of these genes must be produced in the correct stoichiometry and inserted into the membrane, posing a unique challenge to the mitochondrial translational system. In this review we describe the proteins orchestrating mitochondrial translation initiation: bacterial-like general initiation factors mIF2 and mIF3, as well as mitochondria-specific components - mRNA-specific translational activators and mRNA-nonspecific accessory initiation factors. We consider how the fast rate of evolution in these organelles has not only created a system that is divergent from that of its bacterial ancestors, but has led to a huge diversity in lineage specific mechanistic features of mitochondrial translation initiation among eukaryotes.
  •  
29.
  • Mangano, Kyle, et al. (author)
  • Context-based sensing of orthosomycin antibiotics by the translating ribosome
  • 2022
  • In: Nature Chemical Biology. - : Nature Publishing Group. - 1552-4450 .- 1552-4469. ; 18:11, s. 1277-1286
  • Journal article (peer-reviewed)abstract
    • Orthosomycin antibiotics inhibit protein synthesis by binding to the large ribosomal subunit in the tRNA accommodation corridor, which is traversed by incoming aminoacyl-tRNAs. Structural and biochemical studies suggested that orthosomycins block accommodation of any aminoacyl-tRNAs in the ribosomal A-site. However, the mode of action of orthosomycins in vivo remained unknown. Here, by carrying out genome-wide analysis of antibiotic action in bacterial cells, we discovered that orthosomycins primarily inhibit the ribosomes engaged in translation of specific amino acid sequences. Our results reveal that the predominant sites of orthosomycin-induced translation arrest are defined by the nature of the incoming aminoacyl-tRNA and likely by the identity of the two C-terminal amino acid residues of the nascent protein. We show that nature exploits this antibiotic-sensing mechanism for directing programmed ribosome stalling within the regulatory open reading frame, which may control expression of an orthosomycin-resistance gene in a variety of bacterial species. 
  •  
30.
  • Mets, Toomas, et al. (author)
  • Mechanism of phage sensing and restriction by toxin-antitoxin-chaperone systems
  • In: Cell Host and Microbe. - 1934-6069.
  • Journal article (peer-reviewed)abstract
    • Toxin-antitoxins (TAs) are prokaryotic two-gene systems composed of a toxin neutralized by an antitoxin. Toxin-antitoxin-chaperone (TAC) systems additionally include a SecB-like chaperone that stabilizes the antitoxin by recognizing its chaperone addiction (ChAD) element. TACs mediate antiphage defense, but the mechanisms of viral sensing and restriction are unexplored. We identify two Escherichia coli antiphage TAC systems containing host inhibition of growth (HigBA) and CmdTA TA modules, HigBAC and CmdTAC. HigBAC is triggered through recognition of the gpV major tail protein of phage λ. Chaperone HigC recognizes gpV and ChAD via analogous aromatic molecular patterns, with gpV outcompeting ChAD to trigger toxicity. For CmdTAC, the CmdT ADP-ribosyltransferase toxin modifies mRNA to halt protein synthesis and limit phage propagation. Finally, we establish the modularity of TACs by creating a hybrid broad-spectrum antiphage system combining the CmdTA TA warhead with a HigC chaperone phage sensor. Collectively, these findings reveal the potential of TAC systems in broad-spectrum antiphage defense.
  •  
31.
  • Mohamad, Merianne, et al. (author)
  • Sal-type ABC-F proteins : intrinsic and common mediators of pleuromutilin resistance by target protection in staphylococci
  • 2022
  • In: Nucleic Acids Research. - : Oxford University Press. - 0305-1048 .- 1362-4962. ; 50:4, s. 2128-2142
  • Journal article (peer-reviewed)abstract
    • The first member of the pleuromutilin (PLM) class suitable for systemic antibacterial chemotherapy in humans recently entered clinical use, underscoring the need to better understand mechanisms of PLM resistance in disease-causing bacterial genera. Of the proteins reported to mediate PLM resistance in staphylococci, the least-well studied to date is Sal(A), a putative ABC-F NTPase that—by analogy to other proteins of this type—may act to protect the ribosome from PLMs. Here, we establish the importance of Sal proteins as a common source of PLM resistance across multiple species of staphylococci. Sal(A) is revealed as but one member of a larger group of Sal-type ABC-F proteins that vary considerably in their ability to mediate resistance to PLMs and other antibiotics. We find that specific sal genes are intrinsic to particular staphylococcal species, and show that this gene family is likely ancestral to the genus Staphylococcus. Finally, we solve the cryo-EM structure of a representative Sal-type protein (Sal(B)) in complex with the staphylococcal 70S ribosome, revealing that Sal-type proteins bind into the E site to mediate target protection, likely by displacing PLMs and other antibiotics via an allosteric mechanism.
  •  
32.
  • Murina, Victoriia, et al. (author)
  • Antibiotic resistance ABCF proteins reset the peptidyl transferase centre of the ribosome to counter translational arrest
  • 2018
  • In: Nucleic Acids Research. - : Oxford University Press. - 0305-1048 .- 1362-4962. ; 46:7, s. 3753-3763
  • Journal article (peer-reviewed)abstract
    • Several ATPases in the ATP-binding cassette F (ABCF) family confer resistance to macrolides, lincosamides and streptogramins (MLS) antibiotics. MLS are structurally distinct classes, but inhibit a common target: the peptidyl transferase (PTC) active site of the ribosome. Antibiotic resistance (ARE) ABCFs have recently been shown to operate through direct ribosomal protection, but the mechanistic details of this resistance mechanism are lacking. Using a reconstituted translational system, we dissect the molecular mechanism of Staphylococcus haemolyticus VgaA(LC) and Enterococcus faecalis LsaA on the ribosome. We demonstrate that VgaA(LC) is an NTPase that operates as a molecular machine strictly requiring NTP hydrolysis (not just NTP binding) for antibiotic protection. Moreover, when bound to the ribosome in the NTP-bound form, hydrolytically inactive EQ(2) ABCF ARE mutants inhibit peptidyl transferase activity, suggesting a direct interaction between the ABCF ARE and the PTC. The likely structural candidate responsible for antibiotic displacement by wild type ABCF AREs, and PTC inhibition by the EQ(2) mutant, is the extended inter-ABC domain linker region. Deletion of the linker region renders wild type VgaA(LC) inactive in antibiotic protection and the EQ(2) mutant inactive in PTC inhibition.
  •  
33.
  • Palmer, Tracy, et al. (author)
  • A holin/peptidoglycan hydrolase-dependent protein secretion system
  • 2021
  • In: Molecular Microbiology. - : John Wiley & Sons. - 0950-382X .- 1365-2958. ; 115:3, s. 345-355
  • Journal article (peer-reviewed)abstract
    • Gram-negative bacteria have evolved numerous pathways to secrete proteins across their complex cell envelopes. Here, we describe a protein secretion system which uses a holin membrane protein in tandem with a cell wall editing enzyme to mediate the secretion of substrate proteins from the periplasm to the cell exterior. The identity of the cell wall editing enzymes employed was found to vary across biological systems. For instance, the chitinase secretion pathway of Serratia marcescens uses an endopeptidase to facilitate secretion, whereas the secretion of Typhoid toxin in Salmonella enterica serovar Typhi relies on a muramidase. Various families of holins are also predicted to be involved. Genomic analysis indicates that this pathway is conserved and implicated in the secretion of hydrolytic enzymes and toxins for a range of bacteria. The pairing of holins from different families with various types of peptidoglycan hydrolases suggests that this secretion pathway evolved multiple times. We suggest that the complementary bodies of evidence presented is sufficient to propose that the pathway be named the Type 10 Secretion System (TXSS). Potential mechanisms for secretion across the outer membrane are discussed.
  •  
34.
  • Roghanian, Mohammad, et al. (author)
  • (p)ppGpp controls stringent factors by exploiting antagonistic allosteric coupling between catalytic domains
  • 2021
  • In: Molecular Cell. - : Elsevier. - 1097-2765 .- 1097-4164. ; 81:16, s. 3310-3322.e6
  • Journal article (peer-reviewed)abstract
    • 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.
  •  
35.
  • Saha, Chayan Kumar, 1988- (author)
  • Bioinformatics, evolution and revolution in our understanding of toxin-antitoxin systems
  • 2022
  • Doctoral thesis (other academic/artistic)abstract
    • Bacteria experience a wide range of natural challenges during their life cycles, to which they must respond and adapt to live. Under stressed conditions such as amino acid starvation, bacteria slow down their growth mechanism by producing small alarmone nucleotides guanosine pentaphosphate (pppGpp) and tetraphosphate (ppGpp), collectively referred to as (p)ppGpp. Accumulation of (p)ppGpp results in a comprehensive alteration in cellular metabolism. The alarmone (p)ppGpp is produced and degraded by enzymes belonging to the RelA/SpoT Homologue (RSH) protein family, named for their sequence similarity to the RelA and SpoT enzymes of Escherichia coli. The members of the RSH protein family can be classified into long multi-domain RSHs and short single-domain RSHs. Long RSH enzymes such as RelA and SpoT, carry both the (p)ppGpp hydrolysis (HD) domain and (p)ppGpp synthesis (SYNTH) domain in the N-terminal domain enzymatic region (NTD), in combination with additional domains in the C-terminal domain regulatory region (CTD). Short single-domain RSHs can be divided into small alarmone hydrolases (SAHs) carrying the HD domain, and small alarmone synthetases (SASs) that only have the SYNTH domain. At the beginning of my PhD, I studied the diversity of RSH proteins across the tree of life. I identified 35,615 RSH proteins from analyses of 24,072 genomes. I used large-scale phylogenetic analyses to classify the RSH proteins into 13 long RSHs, 11 SAHs and 30 SASs subfamilies. To address why bacteria often carry multiple SASs in the same genome and predict new functions, I developed a computational tool called FlaGs – standing for Flanking Genes – to analyse the conservation of genomic neighbourhoods in large datasets. I also developed a web-based version of FlaGs, called webFlaGs, that is publicly accessible and is used by biologists all over the world. The application of FlaGs to SAS RSHs led to the discovery that multiple SAS subfamilies are encoded in conserved and frequently overlapping two or three-gene architecture, reminiscent of toxin−antitoxin (TA) systems. Five SAS representatives from the FaRel, FaRel2, PhRel, PhRel2 and CapRel subfamilies were experimentally validated as toxins (toxSASs) that are neutralised by the products of six neighbouring antitoxin genes. The toxSAS enzyme FaRel from Cellulomonas marina is encoded as the central gene of a conserved three-gene architecture and acts through the production of nucleotide alarmone ppGpp and its unusual toxic analogue ppApp, causing a significant depletion of ATP and GTP. FaRel toxicity can be countered by both downstream and upstream cognate antitoxins. The latter contains a SAH domain that neutralises toxicity through degradation of ppGpp as well as ppApp. Combining phylogenetic and FlaGs analyses we have discovered that the DUF4065 domain of unknown function is a widely distributed antitoxin domain in putative TA-like operons with dozens of distinct toxic domains. Nine DUF4065-containing antitoxins and their cognate toxins were experimentally validated as TA pairs using toxicity neutralisation assays. These antitoxins form complexes with their diverse cognate toxins. Given the versatility of DUF4065, we have renamed this domain Panacea. We hypothesise that there are multiple hyperpromiscuous antitoxins like Panacea that can be associated with many non-homologous toxin domains, which may also be hyperpromiscuous. Thus, TA systems across all bacteria can be represented as a network of toxin and antitoxin domain combinations, with hyperpromiscuous domains being hubs in the network. To test this and compute a network, I developed a new, iterative version of FlaGs, called NetFlax (standing for Network-FlaGs for toxins and antitoxins), that can identify TA-like gene architectures in an unsupervised manner and generate a toxin-antitoxin domain interaction network. The results from NetFlax verify our strategy since we have rediscovered multiple previously characterised TAs as well as brand new ones. We find that Panacea is unusual but not unique in its hyperpromiscuity and our network indicates the presence of novel hyperpromiscuous domains still to be explored. Our findings also demonstrate how a core network of TAs is evolutionarily linked to various accessory genome systems, including conjugative transfer and phage defence mechanisms. The existing network can potentially be a framework on which future discoveries of the biological role of TAs can be mapped. 
  •  
36.
  • Saha, Chayan Kumar, et al. (author)
  • FlaGs and webFlaGs : discovering novel biology through the analysis of gene neighbourhood conservation
  • 2020
  • In: Bioinformatics. - : Oxford University Press (OUP). - 1367-4803 .- 1367-4811 .- 1460-2059. ; 37:9, s. 1312-1314
  • Journal article (peer-reviewed)abstract
    • Analysis of conservation of gene neighbourhoods over different evolutionary levels is important for understanding operon and gene cluster evolution, and predicting functional associations. Our tool FlaGs (standing for Flanking Genes) takes a list of NCBI protein accessions as input, clusters neighbourhood-encoded proteins into homologous groups using sensitive sequence searching, and outputs a graphical visualization of the gene neighbourhood and its conservation, along with a phylogenetic tree annotated with flanking gene conservation. FlaGs has demonstrated utility for molecular evolutionary analysis, having uncovered a new toxin-antitoxin system in prokaryotes and bacteriophages. The web tool version of FlaGs (webFlaGs) can optionally include a BLASTP search against a reduced RefSeq database to generate an input accession list and analyse neighbourhood conservation within the same run.
  •  
37.
  • Seibt, Henrik, et al. (author)
  • Elevated levels of VCA0117 in response to external signals activates type VI secretion in Vibrio cholerae A1552
  • 2020
  • In: Environmental Microbiology. - : John Wiley & Sons. - 1462-2912 .- 1462-2920. ; 22:10, s. 4409-4423
  • Journal article (other academic/artistic)abstract
    • The type VI nanomachine is critical for Vibrio cholerae to establish infections and to thrive in niches co‐occupied by competing bacteria. The genes for the type VI structural proteins are encoded in one large and two small auxiliary gene clusters. VCA0117 (VasH) – a σ54‐transcriptional activator – is strictly required for functionality of the type VI secretion system since it controls production of the structural protein Hcp. While some strains constitutively produce a functional system, others do not and require specific growth conditions of low temperature and high osmolarity for expression of the type VI machinery. Here, we trace integration of these regulatory signals to the promoter activity of the large gene cluster in which many components of the machinery and VCA0117 itself are encoded. Using in vivo and in vitro assays and variants of VCA0117, we show that activation of the σ54‐promoters of the auxiliary gene clusters by elevated VCA0117 levels are all that is required to overcome the need for specialized growth conditions. We propose a model in which signal integration via the large operon promoter directs otherwise restrictive levels of VCA0117 that ultimately dictates a sufficient supply of Hcp for completion of a functional type VI secretion system.
  •  
38.
  • Starosta, Agata L, et al. (author)
  • A conserved proline triplet in Val-tRNA synthetase and the origin of elongation factor P
  • 2014
  • In: Cell Reports. - : Cell Press. - 2211-1247. ; 9:2, s. 476-483
  • Journal article (peer-reviewed)abstract
    • Bacterial ribosomes stall on polyproline stretches and require the elongation factor P (EF-P) to relieve the arrest. Yet it remains unclear why evolution has favored the development of EF-P rather than selecting against the occurrence of polyproline stretches in proteins. We have discovered that only a single polyproline stretch is invariant across all domains of life, namely a proline triplet in ValS, the tRNA synthetase, that charges tRNA(Val) with valine. Here, we show that expression of ValS in vivo and in vitro requires EF-P and demonstrate that the proline triplet located in the active site of ValS is important for efficient charging of tRNA(Val) with valine and preventing formation of mischarged Thr-tRNA(Val) as well as efficient growth of E. coli in vivo. We suggest that the critical role of the proline triplet for ValS activity may explain why bacterial cells coevolved the EF-P rescue system.
  •  
39.
  • Starosta, Agata L., et al. (author)
  • Translational stalling at polyproline stretches is modulated by the sequence context upstream of the stall site
  • 2014
  • In: Nucleic Acids Research. - : Oxford University Press. - 0305-1048 .- 1362-4962. ; 42:16, s. 10711-10719
  • Journal article (peer-reviewed)abstract
    • The polymerization of amino acids into proteins occurs on ribosomes, with the rate influenced by the amino acids being polymerized. The imino acid proline is a poor donor and acceptor for peptide-bond formation, such that translational stalling occurs when three or more consecutive prolines (PPP) are encountered by the ribosome. In bacteria, stalling at PPP motifs is rescued by the elongation factor P (EF-P). Using SILAC mass spectrometry of Escherichia coli strains, we identified a subset of PPP-containing proteins for which the expression patterns remained unchanged or even appeared up-regulated in the absence of EF-P. Subsequent analysis using in vitro and in vivo reporter assays revealed that stalling at PPP motifs is influenced by the sequence context upstream of the stall site. Specifically, the presence of amino acids such as Cys and Thr preceding the stall site suppressed stalling at PPP motifs, whereas amino acids like Arg and His promoted stalling. In addition to providing fundamental insight into the mechanism of peptide-bond formation, our findings suggest how the sequence context of polyproline-containing proteins can be modulated to maximize the efficiency and yield of protein production.
  •  
40.
  • Svetlov, Maxim S., et al. (author)
  • Peptidyl-tRNA hydrolase is the nascent chain release factor in bacterial ribosome-associated quality control
  • 2024
  • In: Molecular Cell. - 1097-2765. ; 84:4, s. 5-726
  • Journal article (peer-reviewed)abstract
    • Rescuing stalled ribosomes often involves their splitting into subunits. In many bacteria, the resultant large subunits bearing peptidyl-tRNAs are processed by the ribosome-associated quality control (RQC) apparatus that extends the C termini of the incomplete nascent polypeptides with polyalanine tails to facilitate their degradation. Although the tailing mechanism is well established, it is unclear how the nascent polypeptides are cleaved off the tRNAs. We show that peptidyl-tRNA hydrolase (Pth), the known role of which has been to hydrolyze ribosome-free peptidyl-tRNA, acts in concert with RQC factors to release nascent polypeptides from large ribosomal subunits. Dislodging from the ribosomal catalytic center is required for peptidyl-tRNA hydrolysis by Pth. Nascent protein folding may prevent peptidyl-tRNA retraction and interfere with the peptide release. However, oligoalanine tailing makes the peptidyl-tRNA ester bond accessible for Pth-catalyzed hydrolysis. Therefore, the oligoalanine tail serves not only as a degron but also as a facilitator of Pth-catalyzed peptidyl-tRNA hydrolysis.
  •  
41.
  • Svetlov, Maxim S., et al. (author)
  • Structure of Erm-modified 70S ribosome reveals the mechanism of macrolide resistance
  • 2021
  • In: Nature Chemical Biology. - : Nature Publishing Group. - 1552-4450 .- 1552-4469. ; 17:4, s. 412-420
  • Journal article (peer-reviewed)abstract
    • Structural analysis of the A2058-dimethylated and unmethylated 70S ribosome complex alone and in combination with macrolides reveals the role of the desosamine moiety of macrolides in drug binding and resistance. Many antibiotics inhibit bacterial growth by binding to the ribosome and interfering with protein biosynthesis. Macrolides represent one of the most successful classes of ribosome-targeting antibiotics. The main clinically relevant mechanism of resistance to macrolides is dimethylation of the 23S rRNA nucleotide A2058, located in the drug-binding site, a reaction catalyzed by Erm-type rRNA methyltransferases. Here, we present the crystal structure of the Erm-dimethylated 70S ribosome at 2.4 angstrom resolution, together with the structures of unmethylated 70S ribosome functional complexes alone or in combination with macrolides. Altogether, our structural data do not support previous models and, instead, suggest a principally new explanation of how A2058 dimethylation confers resistance to macrolides. Moreover, high-resolution structures of two macrolide antibiotics bound to the unmodified ribosome reveal a previously unknown role of the desosamine moiety in drug binding, laying a foundation for the rational knowledge-based design of macrolides that can overcome Erm-mediated resistance.
  •  
42.
  • Takada, Hiraku, et al. (author)
  • A role for the S4-domain containing protein YlmH in ribosome-associated quality control in Bacillus subtilis
  • In: Nucleic Acids Research. - 1362-4962. ; , s. 1-17
  • Journal article (peer-reviewed)abstract
    • Ribosomes trapped on mRNAs during protein synthesis need to be rescued for the cell to survive. The most ubiquitous bacterial ribosome rescue pathway is trans-translation mediated by tmRNA and SmpB. Genetic inactivation of trans-translation can be lethal, unless ribosomes are rescued by ArfA or ArfB alternative rescue factors or the ribosome-associated quality control (RQC) system, which in Bacillus subtilis involves MutS2, RqcH, RqcP and Pth. Using transposon sequencing in a trans-translation-incompetent B. subtilis strain we identify a poorly characterized S4-domain-containing protein YlmH as a novel potential RQC factor. Cryo-EM structures reveal that YlmH binds peptidyl-tRNA-50S complexes in a position analogous to that of S4-domain-containing protein RqcP, and that, similarly to RqcP, YlmH can co-habit with RqcH. Consistently, we show that YlmH can assume the role of RqcP in RQC by facilitating the addition of poly-alanine tails to truncated nascent polypeptides. While in B. subtilis the function of YlmH is redundant with RqcP, our taxonomic analysis reveals that in multiple bacterial phyla RqcP is absent, while YlmH and RqcH are present, suggesting that in these species YlmH plays a central role in the RQC.
  •  
43.
  • Takada, Hiraku, et al. (author)
  • Expression of Bacillus subtilis ABCF antibiotic resistance factor VmlR is regulated by RNA polymerase pausing, transcription attenuation, translation attenuation and (p)ppGpp
  • 2022
  • In: Nucleic Acids Research. - : Oxford University Press. - 0305-1048 .- 1362-4962. ; 50:11, s. 6174-6189
  • Journal article (peer-reviewed)abstract
    • Since antibiotic resistance is often associated with a fitness cost, bacteria employ multi-layered regulatory mechanisms to ensure that expression of resistance factors is restricted to times of antibiotic challenge. In Bacillus subtilis, the chromosomally-encoded ABCF ATPase VmlR confers resistance to pleuromutilin, lincosamide and type A streptogramin translation inhibitors. Here we show that vmlR expression is regulated by translation attenuation and transcription attenuation mechanisms. Antibiotic-induced ribosome stalling during translation of an upstream open reading frame in the vmlR leader region prevents formation of an anti-antiterminator structure, leading to the formation of an antiterminator structure that prevents intrinsic termination. Thus, transcription in the presence of antibiotic induces vmlR expression. We also show that NusG-dependent RNA polymerase pausing in the vmlR leader prevents leaky expression in the absence of antibiotic. Furthermore, we demonstrate that induction of VmlR expression by compromised protein synthesis does not require the ability of VmlR to rescue the translational defect, as exemplified by constitutive induction of VmlR by ribosome assembly defects. Rather, the specificity of induction is determined by the antibiotic's ability to stall the ribosome on the regulatory open reading frame located within the vmlR leader. Finally, we demonstrate the involvement of (p)ppGpp-mediated signalling in antibiotic-induced VmlR expression.
  •  
44.
  • Takada, Hiraku, et al. (author)
  • Resolution of ribosomal stalling by EF-P and ABCF ATPases YfmR and YkpA/YbiT
  • In: Nucleic Acids Research. - 1362-4962. ; , s. 1-13
  • Journal article (peer-reviewed)abstract
    • Efficiency of protein synthesis on the ribosome is strongly affected by the amino acid composition of the assembled amino acid chain. Challenging sequences include proline-rich motifs as well as highly positively and negatively charged amino acid stretches. Members of the F subfamily of ABC ATPases (ABCFs) have been long hypothesised to promote translation of such problematic motifs. In this study we have applied genetics and reporter-based assays to characterise the four housekeeping ABCF ATPases of Bacillus subtilis: YdiF, YfmM, YfmR/Uup and YkpA/YbiT. We show that YfmR cooperates with the translation factor EF-P that promotes translation of Pro-rich motifs. Simultaneous loss of both YfmR and EF-P results in a dramatic growth defect. Surprisingly, this growth defect can be largely suppressed though overexpression of an EF-P variant lacking the otherwise crucial 5-amino-pentanolylated residue K32. Using in vivo reporter assays, we show that overexpression of YfmR can alleviate ribosomal stalling on Asp-Pro motifs. Finally, we demonstrate that YkpA/YbiT promotes translation of positively and negatively charged motifs but is inactive in resolving ribosomal stalls on proline-rich stretches. Collectively, our results provide insights into the function of ABCF translation factors in modulating protein synthesis in B. subtilis.
  •  
45.
  • Takada, Hiraku, et al. (author)
  • RqcH and RqcP catalyze processive poly-alanine synthesis in a reconstituted ribosome-associated quality control system
  • 2021
  • In: Nucleic Acids Research. - : Oxford University Press. - 0305-1048 .- 1362-4962. ; 49:14, s. 8355-8369
  • Journal article (peer-reviewed)abstract
    • In the cell, stalled ribosomes are rescued through ribosome-associated protein quality-control (RQC) pathways. After splitting of the stalled ribosome, a C-terminal polyalanine 'tail' is added to the unfinished polypeptide attached to the tRNA on the 50S ribosomal subunit. In Bacillus subtilis, polyalanine tailing is catalyzed by the NEMF family protein RqcH, in cooperation with RqcP. However, the mechanistic details of this process remain unclear. Here we demonstrate that RqcH is responsible for tRNAAla selection during RQC elongation, whereas RqcP lacks any tRNA specificity. The ribosomal protein uL11 is crucial for RqcH, but not RqcP, recruitment to the 50S subunit, and B. subtilis lacking uL11 are RQC-deficient. Through mutational mapping, we identify critical residues within RqcH and RqcP that are important for interaction with the P-site tRNA and/or the 50S subunit. Additionally, we have reconstituted polyalanine-tailing in vitro and can demonstrate that RqcH and RqcP are necessary and sufficient for processivity in a minimal system. Moreover, the in vitro reconstituted system recapitulates our in vivo findings by reproducing the importance of conserved residues of RqcH and RqcP for functionality. Collectively, our findings provide mechanistic insight into the role of RqcH and RqcP in the bacterial RQC pathway.
  •  
46.
  • Takada, Hiraku, et al. (author)
  • The C-Terminal RRM/ACT Domain Is Crucial for Fine-Tuning the Activation of 'Long' RelA-SpoT Homolog Enzymes by Ribosomal Complexes
  • 2020
  • In: Frontiers in Microbiology. - : Frontiers Media S.A.. - 1664-302X. ; 11
  • Journal article (peer-reviewed)abstract
    • The (p)ppGpp-mediated stringent response is a bacterial stress response implicated in virulence and antibiotic tolerance. Both synthesis and degradation of the (p)ppGpp alarmone nucleotide are mediated by RelA-SpoT Homolog (RSH) enzymes which can be broadly divided in two classes: single-domain 'short' and multi-domain 'long' RSH. The regulatory ACT (Aspartokinase, Chorismate mutase and TyrA)/RRM (RNA Recognition Motif) domain is a near-universal C-terminal domain of long RSHs. Deletion of RRM in both monofunctional (synthesis-only) RelA as well as bifunctional (i.e., capable of both degrading and synthesizing the alarmone) Rel renders the long RSH cytotoxic due to overproduction of (p)ppGpp. To probe the molecular mechanism underlying this effect we characterized Escherichia coli RelA and Bacillus subtilis Rel RSHs lacking RRM. We demonstrate that, first, the cytotoxicity caused by the removal of RRM is counteracted by secondary mutations that disrupt the interaction of the RSH with the starved ribosomal complex - the ultimate inducer of (p)ppGpp production by RelA and Rel - and, second, that the hydrolytic activity of Rel is not abrogated in the truncated mutant. Therefore, we conclude that the overproduction of (p)ppGpp by RSHs lacking the RRM domain is not explained by a lack of auto-inhibition in the absence of RRM or/and a defect in (p)ppGpp hydrolysis. Instead, we argue that it is driven by misregulation of the RSH activation by the ribosome.
  •  
47.
  • Tamman, Hedvig, et al. (author)
  • Structure of SpoT reveals evolutionary tuning of catalysis via conformational constraint
  • 2023
  • In: Nature Chemical Biology. - : Springer Nature. - 1552-4450 .- 1552-4469. ; 19, s. 334-345
  • Journal article (peer-reviewed)abstract
    • Stringent factors orchestrate bacterial cell reprogramming through increasing the level of the alarmones (p)ppGpp. In Beta- and Gammaproteobacteria, SpoT hydrolyzes (p)ppGpp to counteract the synthetase activity of RelA. However, structural information about how SpoT controls the levels of (p)ppGpp is missing. Here we present the crystal structure of the hydrolase-only SpoT from Acinetobacter baumannii and uncover the mechanism of intramolecular regulation of ‘long’-stringent factors. In contrast to ribosome-associated Rel/RelA that adopt an elongated structure, SpoT assumes a compact τ-shaped structure in which the regulatory domains wrap around a Core subdomain that controls the conformational state of the enzyme. The Core is key to the specialization of long RelA-SpoT homologs toward either synthesis or hydrolysis: the short and structured Core of SpoT stabilizes the τ-state priming the hydrolase domain for (p)ppGpp hydrolysis, whereas the longer, more dynamic Core domain of RelA destabilizes the τ-state priming the monofunctional RelA for efficient (p)ppGpp synthesis. [Figure not available: see fulltext.].
  •  
48.
  • Wilson, Daniel N., et al. (author)
  • Target protection as a key antibiotic resistance mechanism
  • 2020
  • In: Nature Reviews Microbiology. - : Nature Publishing Group. - 1740-1526 .- 1740-1534. ; 18:11, s. 637-648
  • Research review (peer-reviewed)abstract
    • Antibiotic resistance is mediated through several distinct mechanisms, most of which are relatively well understood and the clinical importance of which has long been recognized. Until very recently, neither of these statements was readily applicable to the class of resistance mechanism known as target protection, a phenomenon whereby a resistance protein physically associates with an antibiotic target to rescue it from antibiotic-mediated inhibition. In this Review, we summarize recent progress in understanding the nature and importance of target protection. In particular, we describe the molecular basis of the known target protection systems, emphasizing that target protection does not involve a single, uniform mechanism but is instead brought about in several mechanistically distinct ways.
  •  
49.
  • Zhang, Tong, et al. (author)
  • Direct activation of a bacterial innate immune system by a viral capsid protein
  • 2022
  • In: Nature. - : Springer Science and Business Media LLC. - 0028-0836 .- 1476-4687. ; 612:7938, s. 132-140
  • Journal article (peer-reviewed)abstract
    • Bacteria have evolved diverse immunity mechanisms to protect themselves against the constant onslaught of bacteriophages1-3. Similar to how eukaryotic innate immune systems sense foreign invaders through pathogen-associated molecular patterns4 (PAMPs), many bacterial immune systems that respond to bacteriophage infection require phage-specific triggers to be activated. However, the identities of such triggers and the sensing mechanisms remain largely unknown. Here we identify and investigate the anti-phage function of CapRelSJ46, a fused toxin-antitoxin system that protects Escherichia coli against diverse phages. Using genetic, biochemical and structural analyses, we demonstrate that the C-terminal domain of CapRelSJ46 regulates the toxic N-terminal region, serving as both antitoxin and phage infection sensor. Following infection by certain phages, newly synthesized major capsid protein binds directly to the C-terminal domain of CapRelSJ46 to relieve autoinhibition, enabling the toxin domain to pyrophosphorylate tRNAs, which blocks translation to restrict viral infection. Collectively, our results reveal the molecular mechanism by which a bacterial immune system directly senses a conserved, essential component of phages, suggesting a PAMP-like sensing model for toxin-antitoxin-mediated innate immunity in bacteria. We provide evidence that CapRels and their phage-encoded triggers are engaged in a 'Red Queen conflict'5, revealing a new front in the intense coevolutionary battle between phages and bacteria. Given that capsid proteins of some eukaryotic viruses are known to stimulate innate immune signalling in mammalian hosts6-10, our results reveal a deeply conserved facet of immunity.
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