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The Kluyveromyces lactis γ-toxin targets tRNA anticodons.
Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). (Byström)
Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). (Byström)
Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). (Byström)
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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
Description
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.

Place, publisher, year, edition, pages
2005. Vol. 11, no 11, 1648-1654 p.
Keyword [en]
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: urn:nbn:se:umu:diva-16758DOI: 10.1261/rna.2172105PubMedID: 16244131OAI: oai:DiVA.org:umu-16758DiVA: diva2:156431
Available from: 2007-10-10 Created: 2007-10-10 Last updated: 2017-12-14Bibliographically approved
In thesis
1. The Kluyveromyces lactis killer toxin is a transfer RNA endonuclease
Open this publication in new window or tab >>The Kluyveromyces lactis killer toxin is a transfer RNA endonuclease
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
Molecular biology, K. lactis γ-toxin, tRNA endonuclease, modified nucleosides, 5-methoxycarbonylmethyl-2-thiouridine, 5-methylaminomethyl-2-thiouridine, anticodon stem loop, Molekylärbiologi
Research subject
Molecular Biology
Identifiers
urn:nbn:se:umu:diva-1092 (URN)978-91-7264-303-1 (ISBN)
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
2. Functional aspects of wobble uridine modifications in yeast tRNA
Open this publication in new window or tab >>Functional aspects of wobble uridine modifications in yeast tRNA
2007 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Transfer RNAs (tRNA) function as adaptor molecules in the translation of mRNA into protein. These adaptor molecules require modifications of a subset of their nucleosides for optimal function. The most frequently modified nucleoside in tRNA is position 34 (wobble position), and especially uridines present at this position. Modified nucleosides at the wobble position are important in the decoding process of mRNA, i.e., restriction or improvement of codon-anticodon interactions. This thesis addresses the functional aspects of the wobble uridine modifications.

The Saccharomyces cerevisiae Elongator complex consisting of the six Elp1-Elp6 proteins has been proposed to participate in three distinct cellular processes; elongation of RNA polymerase II transcription, regulation of polarized exocytosis, and formation of modified wobble nucleosides in tRNA. In Paper I, we show that the phenotypes of Elongator deficient cells linking the complex to transcription and exocytosis are counteracted by increased level of and . These tRNAs requires the Elongator complex for formation of the 5-methoxycarbonylmethyl (mcmlnGUUGsmcm25tRNALysUUUsmcm25tRNA5) group of their modified wobble nucleoside 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U). Our results therefore indicate that the relevant function of the Elongator complex is in formation of modified nucleosides in tRNAs and the defects observed in exocytosis and transcription are indirectly caused by inefficient translation of mRNAs encoding gene products important for these processes.

The lack of defined mutants in eukaryotes has led to limited understanding about the role of the wobble uridine modifications in this domain of life. In Paper II, we utilized recently characterized mutants lacking the 2-thio (s2) or 5-carbamoylmethyl (ncm5) and mcm5 groups to address the in vivo function of eukaryotic wobble uridine modifications. We show that ncm5 and mcm5 side-chains promote reading of G-ending codons, and that presence of a mcm5 and an s2 group cooperatively improves reading of both A- and G-ending codons.

Previous studies revealed that a S. cerevisiae strain deleted for any of the six Elongator subunit genes shows resistance towards a toxin (zymocin) secreted by the dairy yeast Kluyveromyces lactis. In Paper III, we show that the cytotoxic γ subunit of zymocin is a tRNA endonuclease that target the anticodon of mcm5s2U34 containing tRNAs and that the wobble mcm5 modification is required for efficient cleavage. This explains the γ-toxin resistant phenotype of Elongator mutants which are defective in the synthesis of the mcm5 group.

Place, publisher, year, edition, pages
Umeå: Molekylärbiologi (Teknisk-naturvetenskaplig fakultet), 2007. 53 p.
Series
Doctoral thesis / Umeå University, Department of Molecular Biology
Keyword
Molecular biology, Transfer RNA, Modified nucleosides, Elongator complex, Translation, Kluyveromyces lactis γ-toxin, Molekylärbiologi
Research subject
Molecular Biology
Identifiers
urn:nbn:se:umu:diva-1093 (URN)978-91-7264-302-4 (ISBN)
Public defence
2007-05-16, Major Groove, 6L, Umeå University, Umeå, 10:00 (English)
Opponent
Supervisors
Available from: 2007-04-23 Created: 2007-04-23 Last updated: 2010-01-08Bibliographically approved

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