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The Kluyveromyces lactis killer toxin is a transfer RNA endonuclease
Umeå University, Faculty of Medicine, Molecular Biology.
2007 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Killer strains of the yeast Kluyveromyces lactis secrete a heterotrimeric protein toxin (zymocin) to inhibit the growth of sensitive yeasts. The cytotoxicity of zymocin resides in the γ subunit (γ-toxin), however the mechanism of cytotoxicity caused by γ-toxin was previously unknown. This thesis aimed to unravel the mode of γ-toxin action and characterize the interaction between γ-toxin and its substrates.

Previous studies suggested a link between the action of γ-toxin and a distinct set of transfer RNAs. In paper I, we show that γ-toxin is a tRNA anticodon endonuclease which cleaves tRNA carrying modified nucleoside 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U) at position 34 (wobble position). The cleavage occurs 3’ to the wobble uridine and yields 2’, 3’-cyclic phosphate and 5´-hydroxyl termini.

In paper II, we identified the determinants in tRNA important for efficient γ-toxin cleavage. The modifications present on the wobble uridines have different effects on tRNA cleavage by γ-toxin. The Saccharomyces cerevisiae wobble modification mcm5 group has a strong positive effect, whereas the Escherichia coli wobble modification 5-methylaminomethyl group has a strong inhibitory effect on tRNA cleavage. The s2 group present in both S. cerevisiae and E. coli tRNAs has a weaker positive effect on the cleavage. The anticodon stem loop (ASL) of tRNA represents the minimal structural requirement for γ-toxin action. Nucleotides U34U35C36A37C38 in the ASL are required for optimal cleavage by γ-toxin, whereas a purine at position 32 or a G at position 33 dramatically reduces the reactivity of ASL.

Screening for S. cerevisiae mutants resistant to zymocin led to the identification of novel proteins important for mcm5s2U formation (paper III). Sit4p (a protein phosphatase), Sap185p and Sap190p (two of the Sit4 associated proteins), and Kti14p (a protein kinase) are required for the formation of mcm5 side chain. Ncs2p, Ncs6p, Urm1p, and Uba4p, the latter two function in a protein modification (urmylation) pathway, are required for the formation of s2 group. The gene product of YOR251C is also involved in the formation of s2 group. The involvement of multiple proteins suggests that the biogenesis of mcm5s2U is very complex.

Place, publisher, year, edition, pages
Umeå: Molekylärbiologi (Teknisk-naturvetenskaplig fakultet) , 2007. , 51 p.
Series
Doctoral thesis / Umeå University, Department of Molecular Biology
Keyword [en]
Molecular biology, K. lactis γ-toxin, tRNA endonuclease, modified nucleosides, 5-methoxycarbonylmethyl-2-thiouridine, 5-methylaminomethyl-2-thiouridine, anticodon stem loop
Keyword [sv]
Molekylärbiologi
Research subject
Molecular Biology
Identifiers
URN: urn:nbn:se:umu:diva-1092ISBN: 978-91-7264-303-1 (print)OAI: oai:DiVA.org:umu-1092DiVA: diva2:140163
Public defence
2007-05-15, Major Groove, 6L, Umeå University, Umeå, 10:00 (English)
Opponent
Supervisors
Available from: 2007-04-23 Created: 2007-04-23 Last updated: 2009-05-19Bibliographically approved
List of papers
1. The Kluyveromyces lactis γ-toxin targets tRNA anticodons.
Open this publication in new window or tab >>The Kluyveromyces lactis γ-toxin targets tRNA anticodons.
Show others...
2005 (English)In: RNA: A publication of the RNA Society, ISSN 1355-8382, E-ISSN 1469-9001, Vol. 11, no 11, 1648-1654 p.Article in journal (Refereed) Published
Abstract [en]

Kluyveromyces lactis killer strains secrete a heterotrimeric toxin (zymocin), which causes an irreversible growth arrest of sensitive yeast cells. Despite many efforts, the target(s) of the cytotoxic gamma-subunit of zymocin has remained elusive. Here we show that three tRNA species tRNA(Glu)(mcm(5)s(2)UUC), tRNA(Lys)(mcm(5)s(2)UUU), and tRNA(Gln)(mcm(5)s(2)UUG) are the targets of gamma-toxin. The toxin inhibits growth by cleaving these tRNAs at the 3' side of the modified wobble nucleoside 5-methoxycarbonylmethyl-2-thiouridine (mcm(5)s(2)U). Transfer RNA lacking a part of or the entire mcm(5) group is inefficiently cleaved by gamma-toxin, explaining the gamma-toxin resistance of the modification-deficient trm9, elp1-elp6, and kti11-kti13 mutants. The K. lactis gamma-toxin is the first eukaryotic toxin shown to target tRNA.

Keyword
Anticodon/*drug effects, Drug Resistance; Fungal/genetics, Gene Expression, Kluyveromyces/*chemistry, Mutation/genetics, Mycotoxins/genetics/*pharmacology, Nucleic Acid Conformation, RNA; Transfer/*drug effects, Saccharomyces cerevisiae/genetics, Saccharomyces cerevisiae Proteins/physiology
Identifiers
urn:nbn:se:umu:diva-16758 (URN)10.1261/rna.2172105 (DOI)16244131 (PubMedID)
Available from: 2007-10-10 Created: 2007-10-10 Last updated: 2017-12-14Bibliographically approved
2. Identification of determinants in tRNA required for Kluyveromyces lactis γ-toxin cleavage.
Open this publication in new window or tab >>Identification of determinants in tRNA required for Kluyveromyces lactis γ-toxin cleavage.
Manuscript (Other academic)
Identifiers
urn:nbn:se:umu:diva-2245 (URN)
Available from: 2007-04-23 Created: 2007-04-23 Last updated: 2010-01-13Bibliographically approved
3. A genome-wide screen in Saccharomyces cerevisiae for mutants defective in the formation of wobble nucleoside 5-methoxycarbonylmethyl-2-thiouridine.
Open this publication in new window or tab >>A genome-wide screen in Saccharomyces cerevisiae for mutants defective in the formation of wobble nucleoside 5-methoxycarbonylmethyl-2-thiouridine.
(English)Manuscript (Other (popular science, discussion, etc.))
Identifiers
urn:nbn:se:umu:diva-2246 (URN)
Available from: 2007-04-23 Created: 2007-04-23 Last updated: 2010-01-14Bibliographically approved

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