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A genome-wide screen in Saccharomyces cerevisiae for mutants defective in the formation of wobble nucleoside 5-methoxycarbonylmethyl-2-thiouridine.
Umeå University, Faculty of Medicine, Molecular Biology.
Umeå University, Faculty of Medicine, Molecular Biology.
(English)Manuscript (Other (popular science, discussion, etc.))
Identifiers
URN: urn:nbn:se:umu:diva-2246OAI: oai:DiVA.org:umu-2246DiVA: diva2:140162
Available from: 2007-04-23 Created: 2007-04-23 Last updated: 2010-01-14Bibliographically 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. Formation and function of wobble uridine modifications in transfer RNA of Saccharomyces cerevisiae
Open this publication in new window or tab >>Formation and function of wobble uridine modifications in transfer RNA of Saccharomyces cerevisiae
2007 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Transfer RNAs (tRNAs) act as adaptor molecules in decoding messenger RNA into protein. Frequently found in tRNAs are different modified nucleosides, which are derivatives of the four normal nucleosides, adenosine (A), guanosine (G), cytidine (C), and uridine (U). Although modified nucleosides are present at many positions in tRNAs, two positions in the anticodon region, position 34 (wobble position) and position 37, show the largest variety of modified nucleosides. In Saccharomyces cerevisiae, the xm5U type of modified uridines found at position 34 are 5-carbamoylmethyluridine (ncm5U), 5-carbamoylmethyl-2´-O-methyluridine, (ncm5Um), 5-methoxycarbonylmethyluridine (mcm5U), and 5-methoxycarbonyl-methyl-2-thiouridine (mcm5s2U). Based on the complex structure of these nucleosides, it is likely that their formation requires several synthesis steps.

The Elongator complex consisting of proteins Elp1p - Elp6p, and the proteins Kti11p - Kti14p, Sit4p, Sap185p, and Sap190p were shown to be involved in 5-carbamoylmethyl (ncm5) and 5-methoxycarbonylmethyl (mcm5) side-chain synthesis at position 34 in eleven tRNA species. The proteins Urm1p, Uba4p, Ncs2p, Ncs6p, and Yor251cp were also identified to be required for the 2-thio (s2) group formation of the modified nucleoside mcm5s2U at wobble position.

Modified nucleosides in the anticodon region of tRNA influence the efficiency and fidelity of translation. The identification of mutants lacking ncm5-, mcm5-, or s2-group at the wobble position allowed the investigation of the in vivo role of these nucleosides in the tRNA decoding process. It was revealed that the presence of ncm5-, mcm5- or s2-group promotes reading of G-ending codons. The concurrent presence of the mcm5- and the s2-groups in the wobble nucleoside mcm5s2U improves reading of A- and G-ending codons, whereas absence of both groups is lethal to the yeast cell.

The Elongator complex was previously proposed to regulate polarized exocytosis and to participate in elongation of RNA polymerase II transcription. The pleiotropic phenotypes observed in Elongator mutants were therefore suggested to be caused by defects in exocytosis and transcription of many genes. Here it is shown that elevated levels of hypomodified tRNALys [mcm5s2UUU] and tRNAGln[mcm5s2UUG] can efficiently suppress these pleiotropic phenotypes, suggesting that the defects in transcription and exocytosis are indirectly caused by inefficient translation of mRNAs encoding proteins important in these processes.

Place, publisher, year, edition, pages
Umeå: Molekylärbiologi (Teknisk-naturvetenskaplig fakultet), 2007. 44 p.
Keyword
Transfer RNA, Modified nucleoside, Elongator complex, Wobble uridine, Decoding
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-1433 (URN)978-91-7264-450-2 (ISBN)
Public defence
2007-12-12, Major Groove, Byggnad 6L, Dept. of Molecular Biology, Umeå University, Umeå, 10:00 (English)
Opponent
Supervisors
Available from: 2007-11-14 Created: 2007-11-14 Last updated: 2009-10-22Bibliographically approved

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