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A conserved modified wobble nucleoside (mcm5s2U) in lysyl-tRNA is required for viability in yeast.
Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
2007 (English)In: RNA, ISSN 1355-8382, Vol. 13, no 8, 1245-55 p.Article in journal (Refereed) Published
Abstract [en]

Transfer RNAs specific for Gln, Lys, and Glu from all organisms (except Mycoplasma) and organelles have a 2-thiouridine derivative (xm(5)s(2)U) as wobble nucleoside. These tRNAs read the A- and G-ending codons in the split codon boxes His/Gln, Asn/Lys, and Asp/Glu. In eukaryotic cytoplasmic tRNAs the conserved constituent (xm(5)-) in position 5 of uridine is 5-methoxycarbonylmethyl (mcm(5)). A protein (Tuc1p) from yeast resembling the bacterial protein TtcA, which is required for the synthesis of 2-thiocytidine in position 32 of the tRNA, was shown instead to be required for the synthesis of 2-thiouridine in the wobble position (position 34). Apparently, an ancient member of the TtcA family has evolved to thiolate U34 in tRNAs of organisms from the domains Eukarya and Archaea. Deletion of the TUC1 gene together with a deletion of the ELP3 gene, which results in the lack of the mcm(5) side chain, removes all modifications from the wobble uridine derivatives of the cytoplasmic tRNAs specific for Gln, Lys, and Glu, and is lethal to the cell. Since excess of the unmodified form of these three tRNAs rescued the double mutant elp3 tuc1, the primary function of mcm(5)s(2)U34 seems to be to improve the efficiency to read the cognate codons rather than to prevent mis-sense errors. Surprisingly, overexpression of the mcm(5)s(2)U-lacking tRNA(Lys) alone was sufficient to restore viability of the double mutant.

Place, publisher, year, edition, pages
2007. Vol. 13, no 8, 1245-55 p.
Keyword [en]
Amino Acid Sequence, Cytoplasm/metabolism, Molecular Sequence Data, Mutation, Nucleosides/chemistry/metabolism, RNA; Transfer; Amino Acyl/chemistry/*metabolism, RNA; Transfer; Gln/chemistry/metabolism, Saccharomyces cerevisiae/chemistry/cytology/genetics/*metabolism, Saccharomyces cerevisiae Proteins/*metabolism, Sequence Alignment
URN: urn:nbn:se:umu:diva-16717DOI: doi:10.1261/rna.558707PubMedID: 17592039OAI: diva2:156390
Available from: 2008-01-04 Created: 2008-01-04 Last updated: 2011-01-11Bibliographically approved
In thesis
1. 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.
Transfer RNA, Modified nucleoside, Elongator complex, Wobble uridine, Decoding
National Category
Biochemistry and Molecular Biology
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)
Available from: 2007-11-14 Created: 2007-11-14 Last updated: 2009-10-22Bibliographically approved

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