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Hauryliuk, Vasili
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Publications (10 of 32) Show all publications
Kasari, V., Pochopien, A. A., Margus, T., Murina, V., Turnbull, K. J., Zhou, Y., . . . Hauryliuk, V. (2019). A role for the Saccharomyces cerevisiae ABCF protein New1 in translation termination/recycling. Nucleic Acids Research, 47(16), 8807-8820
Open this publication in new window or tab >>A role for the Saccharomyces cerevisiae ABCF protein New1 in translation termination/recycling
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2019 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 47, no 16, p. 8807-8820Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
Oxford University Press, 2019
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-164896 (URN)10.1093/nar/gkz600 (DOI)000490576900040 ()31299085 (PubMedID)
Funder
Swedish Research Council, 2017-03783Swedish Research Council, 201504746Swedish Research Council, 2017-04663Ragnar Söderbergs stiftelseThe Kempe Foundations, JCK1627The Kempe Foundations, SMK-1349Magnus Bergvall Foundation, 2017-02098Åke Wiberg Foundation, M14-0207EU, Horizon 2020, 2643Swedish Research Council, 2017-03783
Available from: 2019-11-05 Created: 2019-11-05 Last updated: 2019-11-07
Murina, V., Kasari, M., Takada, H., Hinnu, M., Kumar Saha, C., Grimshaw, J. W., . . . Atkinson, G. C. (2019). ABCF ATPases Involved in Protein Synthesis, Ribosome Assembly and Antibiotic Resistance: Structural and Functional Diversification across the Tree of Life. Journal of Molecular Biology, 431(18), 3568-3590
Open this publication in new window or tab >>ABCF ATPases Involved in Protein Synthesis, Ribosome Assembly and Antibiotic Resistance: Structural and Functional Diversification across the Tree of Life
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2019 (English)In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 431, no 18, p. 3568-3590Article in journal (Refereed) Published
Abstract [en]

Within the larger ABC superfamily of ATPases, ABCF family members eEF3 in Saccharomyces cerevisiae and EttA in Escherichia coli have been found to function as ribosomal translation factors. Several other ABCFs including biochemically characterized VgaA, LsaA and MsrE confer resistance to antibiotics that target the peptidyl transferase center and exit tunnel of the ribosome. However, the diversity of ABCF subfamilies, the relationships among subfamilies and the evolution of antibiotic resistance (ARE) factors from other ABCFs have not been explored. To address this, we analyzed the presence of ABCFs and their domain architectures in 4505 genomes across the tree of life. We find 45 distinct subfamilies of ABCFs that are widespread across bacterial and eukaryotic phyla, suggesting that they were present in the last common ancestor of both. Surprisingly, currently known ARE ABCFs are not confined to a distinct lineage of the ABCF family tree, suggesting that ARE can readily evolve from other ABCF functions. Our data suggest that there are a number of previously unidentified ARE ABCFs in antibiotic producers and important human pathogens. We also find that ATPase-deficient mutants of all four E. coli ABCFs (EttA, YbiT, YheS and Uup) inhibit protein synthesis, indicative of their ribosomal function, and demonstrate a genetic interaction of ABCFs Uup and YheS with translational GTPase BipA involved in assembly of the 50S ribosome subunit. Finally, we show that the ribosome-binding resistance factor VmlR from Bacillus subtilis is localized to the cytoplasm, ruling out a role in antibiotic efflux.

Place, publisher, year, edition, pages
Elsevier, 2019
Keywords
ribosome, translation, antibiotic resistance, ABCF, ARE
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-164649 (URN)10.1016/j.jmb.2018.12.013 (DOI)000484872800012 ()30597160 (PubMedID)2-s2.0-85060093624 (Scopus ID)
Available from: 2019-10-25 Created: 2019-10-25 Last updated: 2019-10-25Bibliographically approved
Zbornikova, E., Knejzlik, Z., Hauryliuk, V., Krasny, L. & Rejman, D. (2019). Analysis of nucleotide pools in bacteria using HPLC-MS in HILIC mode. Talanta: The International Journal of Pure and Applied Analytical Chemistry, 205, Article ID UNSP 120161.
Open this publication in new window or tab >>Analysis of nucleotide pools in bacteria using HPLC-MS in HILIC mode
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2019 (English)In: Talanta: The International Journal of Pure and Applied Analytical Chemistry, ISSN 0039-9140, E-ISSN 1873-3573, Vol. 205, article id UNSP 120161Article in journal (Refereed) Published
Abstract [en]

Nucleotides, nucleosides and their derivatives are present in all cells at varying concentrations that change with the nutritional, and energetic status of the cell. Precise measurement of the concentrations of these molecules is instrumental for understanding their regulatory effects. Such measurement is challenging due to the inherent instability of these molecules and, despite many decades of research, the reported values differ widely. Here, we present a comprehensive and easy-to-use approach for determination of the intracellular concentrations of > 25 target molecular species. The approach uses rapid filtration and cold acidic extraction followed by high performance liquid chromatography (HPLC) in the hydrophilic interaction liquid chromatography (HILIC) mode using zwitterionic columns coupled with UV and MS detectors. The method reliably detects and quantifies all the analytes expected to be observed in the bacterial cell and paves the way for future studies correlating their concentrations with biological effects.

Place, publisher, year, edition, pages
Elsevier, 2019
Keywords
Nucleotide, HPLC-MS, HILIC, ppGpp, Stringent response, Escherichia coli
National Category
Analytical Chemistry
Identifiers
urn:nbn:se:umu:diva-164375 (URN)10.1016/j.talanta.2019.120161 (DOI)000485856500085 ()31450400 (PubMedID)
Available from: 2019-11-12 Created: 2019-11-12 Last updated: 2019-11-12Bibliographically approved
Turnbull, K. J., Dzhygyr, I., Lindemose, S., Hauryliuk, V. & Roghanian, M. (2019). Intramolecular Interactions Dominate the Autoregulation of Escherichia coli Stringent Factor RelA. Frontiers in Microbiology, 10, Article ID 1966.
Open this publication in new window or tab >>Intramolecular Interactions Dominate the Autoregulation of Escherichia coli Stringent Factor RelA
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2019 (English)In: Frontiers in Microbiology, ISSN 1664-302X, E-ISSN 1664-302X, Vol. 10, article id 1966Article in journal (Refereed) Published
Abstract [en]

Amino acid starvation in Escherichia coli activates the enzymatic activity of the stringent factor RelA, leading to accumulation of the alarmone nucleotide (p)ppGpp. The alarmone acts as an intercellular messenger to regulate transcription, translation and metabolism to mediate bacterial stress adaptation. The enzymatic activity of RelA is subject to multi-layered allosteric control executed both by ligands - such as "starved" ribosomal complexes, deacylated tRNA and pppGpp - and by individual RelA domains. The auto-regulation of RelA is proposed to act either in cis (inhibition of the enzymatic activity of the N-terminal region, NTD, by regulatory C-terminal region, CTD) or in trans (CTD-mediated dimerization leading to enzyme inhibition). In this report, we probed the regulatory roles of the individual domains of E. coli RelA and our results are not indicative of RelA dimerization being the key regulatory mechanism. First, at growth-permitting levels, ectopic expression of RelA CTD does not interfere with activation of native ReIA, indicating lack of regulation via inhibitory complex formation in the cell. Second, in our biochemical assays, increasing RelA concentration does not decrease the enzyme activity, as would be expected in the case of efficient auto-inhibition via dimerization. Third, while high-level CTD expression efficiently inhibits the growth, the effect is independent of native RelA and is mediated by direct inhibition of protein synthesis, likely via direct interaction with the ribosomal A-site. Finally, deletion of the RRM domain of the CTD region leads to growth inhibition mediated by accumulation of (p)ppGpp, suggesting de-regulation of the synthetic activity in this mutant.

Place, publisher, year, edition, pages
Frontiers Media S.A., 2019
Keywords
(p)ppGpp, RelA, RSH, stringent response, ribosome, translation
National Category
Biochemistry and Molecular Biology Medical Biotechnology
Identifiers
urn:nbn:se:umu:diva-163667 (URN)10.3389/fmicb.2019.01966 (DOI)000482805400001 ()
Available from: 2019-10-21 Created: 2019-10-21 Last updated: 2019-10-21Bibliographically approved
Kaldalu, N., Maiväli, Ü., Hauryliuk, V. & Tenson, T. (2019). Reanalysis of Proteomics Results Fails To Detect MazF-Mediated Stress Proteins [Letter to the editor]. mBio, 10(3), Article ID e00949-19.
Open this publication in new window or tab >>Reanalysis of Proteomics Results Fails To Detect MazF-Mediated Stress Proteins
2019 (English)In: mBio, ISSN 2161-2129, E-ISSN 2150-7511, Vol. 10, no 3, article id e00949-19Article in journal, Letter (Refereed) Published
Place, publisher, year, edition, pages
American Society for Microbiology, 2019
Keywords
endonuclease, proteomics, statistics, toxin/antitoxin systems
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-161744 (URN)10.1128/mBio.00949-19 (DOI)000473596500004 ()31186323 (PubMedID)
Available from: 2019-07-25 Created: 2019-07-25 Last updated: 2019-07-25Bibliographically approved
Kasari, V., Margus, T., Atkinson, G. C., Johansson, M. J. O. & Hauryliuk, V. (2019). Ribosome profiling analysis of eEF3-depleted Saccharomyces cerevisiae. Scientific Reports, 9, Article ID 3037.
Open this publication in new window or tab >>Ribosome profiling analysis of eEF3-depleted Saccharomyces cerevisiae
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2019 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 9, article id 3037Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
Nature Publishing Group, 2019
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-157582 (URN)10.1038/s41598-019-39403-y (DOI)000459891700047 ()30816176 (PubMedID)
Available from: 2019-03-29 Created: 2019-03-29 Last updated: 2019-03-29Bibliographically approved
Van Nerom, K., Tamman, H., Takada, H., Hauryliuk, V. & Garcia-Pino, A. (2019). The Rel stringent factor from Thermus thermophilus: crystallization and X-ray analysis. Acta Crystallographica Section F : Structural Biology Communications, 75, 561-569
Open this publication in new window or tab >>The Rel stringent factor from Thermus thermophilus: crystallization and X-ray analysis
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2019 (English)In: Acta Crystallographica Section F : Structural Biology Communications, ISSN 2053-230X, Vol. 75, p. 561-569Article in journal (Refereed) Published
Abstract [en]

The stringent response, controlled by (p)ppGpp, enables bacteria to trigger a strong phenotypic resetting that is crucial to cope with adverse environmental changes and is required for stress survival and virulence. In the bacterial cell, (p)ppGpp levels are regulated by the concerted opposing activities of RSH (RelA/SpoT homologue) enzymes that can transfer a pyrophosphate group of ATP to the 3′ position of GDP (or GTP) or remove the 3′ pyrophosphate moiety from (p)ppGpp. Bifunctional Rel enzymes are notoriously difficult to crystallize owing to poor stability and a propensity for aggregation, usually leading to a loss of biological activity after purification. Here, the production, biochemical analysis and crystallization of the bifunctional catalytic region of the Rel stringent factor from Thermus thermophilus (RelTtNTD) in the resting state and bound to nucleotides are described. RelTt and RelTtNTD are monomers in solution that are stabilized by the binding of Mn2+ and mellitic acid. RelTtNTD crystallizes in space group P4122, with unit-cell parameters a = b = 88.4, c = 182.7 Å, at 4°C and in space group P41212, with unit-cell parameters a = b = 105.7, c = 241.4 Å, at 20°C.

Place, publisher, year, edition, pages
International Union of Crystallography, 2019
Keywords
stringent response, Rel/RelA/SpoT, (p)ppGpp, bacterial alarmone, Thermus thermophilus
National Category
Microbiology in the medical area
Identifiers
urn:nbn:se:umu:diva-162870 (URN)10.1107/S2053230X19010628 (DOI)000480337300007 ()31397328 (PubMedID)
Available from: 2019-09-05 Created: 2019-09-05 Last updated: 2019-09-05Bibliographically approved
Murina, V., Kasari, M., Hauryliuk, V. & Atkinson, G. C. (2018). Antibiotic resistance ABCF proteins reset the peptidyl transferase centre of the ribosome to counter translational arrest. Nucleic Acids Research, 46(7), 3753-3763
Open this publication in new window or tab >>Antibiotic resistance ABCF proteins reset the peptidyl transferase centre of the ribosome to counter translational arrest
2018 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 46, no 7, p. 3753-3763Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
Oxford University Press, 2018
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-147824 (URN)10.1093/nar/gky050 (DOI)000431137900041 ()29415157 (PubMedID)
Available from: 2018-05-18 Created: 2018-05-18 Last updated: 2018-06-09Bibliographically approved
Brodiazhenko, T., Johansson, M. J. O., Takada, H., Nissan, T., Hauryliuk, V. & Murina, V. (2018). Elimination of Ribosome Inactivating Factors Improves the Efficiency of Bacillus subtilis and Saccharomyces cerevisiae Cell-Free Translation Systems. Frontiers in Microbiology, 9, Article ID 3041.
Open this publication in new window or tab >>Elimination of Ribosome Inactivating Factors Improves the Efficiency of Bacillus subtilis and Saccharomyces cerevisiae Cell-Free Translation Systems
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2018 (English)In: Frontiers in Microbiology, ISSN 1664-302X, E-ISSN 1664-302X, Vol. 9, article id 3041Article in journal (Refereed) Published
Abstract [en]

Cell-free translation systems based on cellular lysates optimized for in vitro protein synthesis have multiple applications both in basic and applied science, ranging from studies of translational regulation to cell-free production of proteins and ribosome-nascent chain complexes. In order to achieve both high activity and reproducibility in a translation system, it is essential that the ribosomes in the cellular lysate are enzymatically active. Here we demonstrate that genomic disruption of genes encoding ribosome inactivating factors – HPF in Bacillus subtilis and Stm1 in Saccharomyces cerevisiae – robustly improve the activities of bacterial and yeast translation systems. Importantly, the elimination of B. subtilis HPF results in a complete loss of 100S ribosomes, which otherwise interfere with disome-based approaches for preparation of stalled ribosomal complexes for cryo-electron microscopy studies.

Place, publisher, year, edition, pages
Frontiers Media S.A., 2018
Keywords
HPF, Stm1, Bacillus subtilis, Saccharomyces cerevisiae, cell-tree translation system
National Category
Microbiology
Identifiers
urn:nbn:se:umu:diva-155100 (URN)10.3389/fmicb.2018.03041 (DOI)000453653000001 ()
Funder
Swedish Research Council, 2013-4680Swedish Research Council, 2017-04663Ragnar Söderbergs stiftelseMagnus Bergvall Foundation, 2017-02098Åke Wiberg Foundation, M14-0207
Available from: 2019-01-10 Created: 2019-01-10 Last updated: 2019-01-10Bibliographically approved
Goormaghtigh, F., Fraikin, N., Putrins, M., Hallaert, T., Hauryliuk, V., Garcia-Pino, A., . . . Van Melderen, L. (2018). Reassessing the Role of Type II Toxin-Antitoxin Systems in Formation of Escherichia coli Type II Persister Cells. mBio, 9(3), Article ID e00640-18.
Open this publication in new window or tab >>Reassessing the Role of Type II Toxin-Antitoxin Systems in Formation of Escherichia coli Type II Persister Cells
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2018 (English)In: mBio, ISSN 2161-2129, E-ISSN 2150-7511, Vol. 9, no 3, article id e00640-18Article in journal (Refereed) Published
Abstract [en]

Persistence is a reversible and low-frequency phenomenon allowing a subpopulation of a clonal bacterial population to survive antibiotic treatments. Upon removal of the antibiotic, persister cells resume growth and give rise to viable progeny. Type II toxin-antitoxin (TA) systems were assumed to play a key role in the formation of persister cells in Escherichia coli based on the observation that successive deletions of TA systems decreased persistence frequency. In addition, the model proposed that stochastic fluctuations of (p)ppGpp levels are the basis for triggering activation of TA systems. Cells in which TA systems are activated are thought to enter a dormancy state and therefore survive the antibiotic treatment. Using independently constructed strains and newly designed fluorescent reporters, we reassessed the roles of TA modules in persistence both at the population and single-cell levels. Our data confirm that the deletion of 10 TA systems does not affect persistence to ofloxacin or ampicillin. Moreover, microfluidic experiments performed with a strain reporting the induction of the yefM-yoeB TA system allowed the observation of a small number of type II persister cells that resume growth after removal of ampicillin. However, we were unable to establish a correlation between high fluorescence and persistence, since the fluorescence of persister cells was comparable to that of the bulk of the population and none of the cells showing high fluorescence were able to resume growth upon removal of the antibiotic. Altogether, these data show that there is no direct link between induction of TA systems and persistence to antibiotics. IMPORTANCE Within a growing bacterial population, a small subpopulation of cells is able to survive antibiotic treatment by entering a transient state of dormancy referred to as persistence. Persistence is thought to be the cause of relapsing bacterial infections and is a major public health concern. Type II toxin-antitoxin systems are small modules composed of a toxic protein and an antitoxin protein counteracting the toxin activity. These systems were thought to be pivotal players in persistence until recent developments in the field. Our results demonstrate that previous influential reports had technical flaws and that there is no direct link between induction of TA systems and persistence to antibiotics.

Place, publisher, year, edition, pages
American Society for Microbiology, 2018
Keywords
RelE, YoeB, ampicillin, single cell
National Category
Biological Sciences
Identifiers
urn:nbn:se:umu:diva-155657 (URN)10.1128/mBio.00640-18 (DOI)000454748900022 ()29895634 (PubMedID)
Available from: 2019-01-25 Created: 2019-01-25 Last updated: 2019-01-25Bibliographically approved
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