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PMID: 10864043 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

Identification of the TRM2 gene encoding the tRNA(m5U54)methyltransferase of Saccharomyces cerevisiae.

RNA (New York, N.Y.) ·Vol. 6 ·No. 6 ·2000-06-00 ·Pages 844-60

Nordlund ME, Johansson JO, von Pawel-Rammingen U, Byström AS

Abstract

The presence of 5-methyluridine (m5U) at position 54 is a ubiquitous feature of most bacterial and eukaryotic elongator tRNAs. In this study, we have identified and characterized the TRM2 gene that encodes the tRNA(m5U54)methyltransferase, responsible for the formation of this modified nucleoside in Saccharomyces cerevisiae. Transfer RNA isolated from TRM2-disrupted yeast strains does not contain the m5U54 nucleoside. Moreover, a glutathione S-transferase (GST) tagged recombinant, Trm2p, expressed in Escherichia coli displayed tRNA(m5U54)methyltransferase activity using as substrate tRNA isolated from a trm2 mutant strain, but not tRNA isolated from a TRM2 wild-type strain. In contrast to what is found for the tRNA(m5U54)methyltransferase encoding gene trmA+ in E. coli, the TRM2 gene is not essential for cell viability and a deletion strain shows no obvious phenotype. Surprisingly, we found that the TRM2 gene was previously identified as the RNC1/NUD1 gene, believed to encode the yNucR endo-exonuclease. The expression and activity of the yNucR endo-exonuclease is dependent on the RAD52 gene, and does not respond to increased gene dosage of the RNC1/NUD1 gene. In contrast, we find that the expression of a trm2-LacZ fusion and the activity of the tRNA(m5U54)methyltransferase is not regulated by the RAD52 gene and does respond on increased gene dosage of the TRM2 (RNC1/NUD1) gene. Furthermore, there was no nuclease activity associated with a GST-Trm2 recombinant protein. The purified yNucR endo-exonuclease has been reported to have an NH2-D-E-K-N-L motif, which is not found in the Trm2p. Therefore, we suggest that the yNucR endo-exonuclease is encoded by a gene other than TRM2.

MeSH Terms
3' Untranslated Regions/genetics 5' Untranslated Regions/genetics Amino Acid Sequence Base Sequence DNA, Fungal/metabolism DNA-Binding Proteins/metabolism Deoxyribonucleases/genetics Escherichia coli/enzymology,genetics Fungal Proteins/genetics,isolation & purification Gene Dosage Genes, Fungal Molecular Sequence Data Mutation/genetics Rad52 DNA Repair and Recombination Protein Saccharomyces cerevisiae/enzymology,genetics Saccharomyces cerevisiae Proteins tRNA Methyltransferases/genetics,isolation & purification,physiology
Chemicals
3' Untranslated Regions 5' Untranslated Regions DNA, Fungal DNA-Binding Proteins Fungal Proteins RAD52 protein, S cerevisiae Rad52 DNA Repair and Recombination Protein Saccharomyces cerevisiae Proteins tRNA Methyltransferases tRNA(uracil-5)-methyltransferase Deoxyribonucleases TRM2 protein, S cerevisiae
Authors & Affiliations
4 authors, click to expand affiliations / ORCID
Nordlund M E
Department of Microbiology, Umeå University, Sweden.
Johansson J O
von Pawel-Rammingen U
Byström A S
References (65)
65 references, click to expand
  1. BLAST 2 Sequences, a new tool for comparing protein and nucleotide sequences.
    FEMS Microbiol Lett. 1999 May 15;174(2):247-50 PMID: 10339815
  2. An adenosine deaminase that generates inosine at the wobble position of tRNAs.
    Science. 1999 Nov 5;286(5442):1146-9 PMID: 10550050
  3. A convenient procedure for preparing transfer ribonucleic acid from Escherichia coli.
    Biochim Biophys Acta. 1969 Apr 22;179(2):297-307 PMID: 4890595
  4. Physiological and biochemical studies on the function of 5-methyluridine in the transfer ribonucleic acid of Escherichia coli.
    J Bacteriol. 1975 Oct;124(1):99-111 PMID: 1100618
  5. Amino acid sequence at the FdUMP binding site of thymidylate synthetase.
    Proc Natl Acad Sci U S A. 1976 Jun;73(6):1848-52 PMID: 1064857
  6. DNA sequencing with chain-terminating inhibitors.
    Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463-7 PMID: 271968
  7. Replacement of chromosome segments with altered DNA sequences constructed in vitro.
    Proc Natl Acad Sci U S A. 1979 Oct;76(10):4951-5 PMID: 388424
  8. The RAD52 gene is required for homothallic interconversion of mating types and spontaneous mitotic recombination in yeast.
    Proc Natl Acad Sci U S A. 1980 Jan;77(1):503-7 PMID: 6987653
  9. On the role of ribosylthymine in prokaryotic tRNA function.
    Eur J Biochem. 1981 Feb;114(2):451-6 PMID: 7011814
  10. Yeast transformation: a model system for the study of recombination.
    Proc Natl Acad Sci U S A. 1981 Oct;78(10):6354-8 PMID: 6273866
  11. Two differentially regulated mRNAs with different 5' ends encode secreted with intracellular forms of yeast invertase.
    Cell. 1982 Jan;28(1):145-54 PMID: 7039847
  12. Defects in modification of cytoplasmic and mitochondrial transfer RNAs are caused by single nuclear mutations.
    Cell. 1982 Mar;28(3):543-50 PMID: 7074684
  13. Quantitative enzymatic hydrolysis of tRNAs: reversed-phase high-performance liquid chromatography of tRNA nucleosides.
    J Chromatogr. 1982 Jul 9;230(2):297-308 PMID: 7050138
  14. Complete analysis of tRNA-modified nucleosides by high-performance liquid chromatography: the 29 modified nucleosides of Salmonella typhimurium and Escherichia coli tRNA.
    Anal Biochem. 1983 Feb 15;129(1):1-13 PMID: 6190418
  15. Purification and properties of single strand DNA-binding endo-exonuclease of Neurospora crassa.
    J Biol Chem. 1983 Oct 10;258(19):12010-8 PMID: 6311833
  16. Rapid and efficient site-specific mutagenesis without phenotypic selection.
    Proc Natl Acad Sci U S A. 1985 Jan;82(2):488-92 PMID: 3881765
  17. Ty elements transpose through an RNA intermediate.
    Cell. 1985 Mar;40(3):491-500 PMID: 2982495
  18. DNA polymerases, deoxyribonucleases, and recombination during meiosis in Saccharomyces cerevisiae.
    Mol Cell Biol. 1984 Dec;4(12):2811-7 PMID: 6396507
  19. Isolation and characterization of the TRM1 locus, a gene essential for the N2,N2-dimethylguanosine modification of both mitochondrial and cytoplasmic tRNA in Saccharomyces cerevisiae.
    J Biol Chem. 1986 Jul 25;261(21):9703-9 PMID: 2426253
  20. Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes.
    J Mol Biol. 1986 May 5;189(1):113-30 PMID: 3537305
  21. Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase.
    Gene. 1988 Jul 15;67(1):31-40 PMID: 3047011
  22. 5-Fluorocytosine in DNA is a mechanism-based inhibitor of HhaI methylase.
    Biochemistry. 1988 Jul 12;27(14):5204-10 PMID: 3167042
  23. Yeast/E. coli shuttle vectors with multiple unique restriction sites.
    Yeast. 1986 Sep;2(3):163-7 PMID: 3333305
  24. The scanning model for translation: an update.
    J Cell Biol. 1989 Feb;108(2):229-41 PMID: 2645293
  25. A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae.
    Genetics. 1989 May;122(1):19-27 PMID: 2659436
  26. A functional analysis of the repeated methionine initiator tRNA genes (IMT) in yeast.
    Mol Gen Genet. 1989 Apr;216(2-3):276-86 PMID: 2664453
  27. Neurospora endo-exonuclease is immunochemically related to the recC gene product of Escherichia coli.
    J Bacteriol. 1990 Jan;172(1):507-10 PMID: 2152915
  28. Upstream activating sequences that are shared by two divergently transcribed operons mediate cAMP-CRP regulation of pilus-adhesin in Escherichia coli.
    Mol Microbiol. 1989 Nov;3(11):1557-65 PMID: 2575704
  29. High-level expression of Escherichia coli tRNA (m5U54)-methyltransferase.
    DNA Cell Biol. 1990 May;9(4):273-8 PMID: 2190591
  30. The trmA promoter has regulatory features and sequence elements in common with the rRNA P1 promoter family of Escherichia coli.
    J Bacteriol. 1991 Mar;173(5):1757-64 PMID: 1999392
  31. Analysis of RNA hydrolyzates by liquid chromatography-mass spectrometry.
    Methods Enzymol. 1990;193:796-824 PMID: 1706064
  32. Putting the HO gene to work: practical uses for mating-type switching.
    Methods Enzymol. 1991;194:132-46 PMID: 2005783
  33. Identification of the catalytic nucleophile of tRNA (m5U54)methyltransferase.
    Biochemistry. 1991 Oct 8;30(40):9724-8 PMID: 1911760
  34. Evidence that an endo-exonuclease controlled by the NUC2 gene functions in the induction of 'petite' mutations in Saccharomyces cerevisiae.
    Curr Genet. 1991 Jul;20(1-2):39-44 PMID: 1657413
  35. Mutational analysis of conserved positions potentially important for initiator tRNA function in Saccharomyces cerevisiae.
    Mol Cell Biol. 1992 Apr;12(4):1432-42 PMID: 1549105
  36. Improved method for high efficiency transformation of intact yeast cells.
    Nucleic Acids Res. 1992 Mar 25;20(6):1425 PMID: 1561104
  37. The gene for a tRNA modifying enzyme, m5U54-methyltransferase, is essential for viability in Escherichia coli.
    Proc Natl Acad Sci U S A. 1992 May 1;89(9):3995-8 PMID: 1373891
  38. Yeast RNC1 encodes a chimeric protein, RhoNUC, with a human rho motif and deoxyribonuclease activity.
    Nucleic Acids Res. 1992 Oct 11;20(19):5215-21 PMID: 1408836
  39. Multisite-specific tRNA:m5C-methyltransferase (Trm4) in yeast Saccharomyces cerevisiae: identification of the gene and substrate specificity of the enzyme.
    RNA. 1999 Aug;5(8):1105-18 PMID: 10445884
  40. Yeast shuttle and integrative vectors with multiple cloning sites suitable for construction of lacZ fusions.
    Gene. 1986;45(3):299-310 PMID: 3026915
  41. Kinetic and catalytic mechanism of HhaI methyltransferase.
    J Biol Chem. 1987 Apr 5;262(10):4778-86 PMID: 3558369
  42. Isolation and characterization of MOD5, a gene required for isopentenylation of cytoplasmic and mitochondrial tRNAs of Saccharomyces cerevisiae.
    Mol Cell Biol. 1987 Jan;7(1):177-84 PMID: 3031456
  43. Purification and characterization of an endo-exonuclease from Saccharomyces cerevisiae that is influenced by the RAD52 gene.
    J Biol Chem. 1987 Dec 25;262(36):17659-67 PMID: 2826428
  44. An endo-exonuclease activity of yeast that requires a functional RAD52 gene.
    Mol Gen Genet. 1988 Jan;211(1):41-8 PMID: 2830467
  45. Separate information required for nuclear and subnuclear localization: additional complexity in localizing an enzyme shared by mitochondria and nuclei.
    Mol Cell Biol. 1992 Dec;12(12):5652-8 PMID: 1448094
  46. The yeast initiator tRNAMet can act as an elongator tRNA(Met) in vivo.
    J Mol Biol. 1993 Sep 5;233(1):43-58 PMID: 8377191
  47. The RHO4a and NUD1 genes on Saccharomyces cerevisiae chromosome XI.
    Yeast. 1993 Oct;9(10):1139-47 PMID: 8256523
  48. Widespread occurrence of three sequence motifs in diverse S-adenosylmethionine-dependent methyltransferases suggests a common structure for these enzymes.
    Arch Biochem Biophys. 1994 May 1;310(2):417-27 PMID: 8179327
  49. Heterogeneous functional Ty1 elements are abundant in the Saccharomyces cerevisiae genome.
    Genetics. 1994 Apr;136(4):1245-59 PMID: 8013902
  50. Rit1, a tRNA backbone-modifying enzyme that mediates initiator and elongator tRNA discrimination.
    Cell. 1994 Nov 4;79(3):535-46 PMID: 7954819
  51. Enzymatic mechanism of tRNA (m5U54)methyltransferase.
    Biochimie. 1994;76(12):1133-42 PMID: 7748948
  52. Redundancy of Saccharomyces cerevisiae MSH3 and MSH6 in MSH2-dependent mismatch repair.
    Genes Dev. 1996 Feb 15;10(4):407-20 PMID: 8600025
  53. Nuclear pore proteins are involved in the biogenesis of functional tRNA.
    EMBO J. 1996 May 1;15(9):2270-84 PMID: 8641292
  54. A Rad52 homolog is required for RAD51-independent mitotic recombination in Saccharomyces cerevisiae.
    Genes Dev. 1996 Aug 15;10(16):2025-37 PMID: 8769646
  55. Comparative mutagenesis of nuclear localization signals reveals the importance of neutral and acidic amino acids.
    Curr Biol. 1996 Aug 1;6(8):1025-7 PMID: 8805337
  56. Exonuclease I of Saccharomyces cerevisiae functions in mitotic recombination in vivo and in vitro.
    Mol Cell Biol. 1997 May;17(5):2764-73 PMID: 9111347
  57. The yeast gene YNL292w encodes a pseudouridine synthase (Pus4) catalyzing the formation of psi55 in both mitochondrial and cytoplasmic tRNAs.
    Nucleic Acids Res. 1997 Nov 15;25(22):4493-9 PMID: 9358157
  58. Characterization of yeast protein Deg1 as pseudouridine synthase (Pus3) catalyzing the formation of psi 38 and psi 39 in tRNA anticodon loop.
    J Biol Chem. 1998 Jan 16;273(3):1316-23 PMID: 9430663
  59. The transcriptional program of sporulation in budding yeast.
    Science. 1998 Oct 23;282(5389):699-705 PMID: 9784122
  60. The essential Gcd10p-Gcd14p nuclear complex is required for 1-methyladenosine modification and maturation of initiator methionyl-tRNA.
    Genes Dev. 1998 Dec 1;12(23):3650-62 PMID: 9851972
  61. Genetic analysis of the yeast NUD1 endo-exonuclease: a role in the repair of DNA double-strand breaks.
    Curr Genet. 1998 Dec;34(5):360-7 PMID: 9871117
  62. Saccharomyces cerevisiae Mod5p-II contains sequences antagonistic for nuclear and cytosolic locations.
    Genetics. 1999 Jan;151(1):57-75 PMID: 9872948
  63. S-Adenosylmethionine-dependent methylation in Saccharomyces cerevisiae. Identification of a novel protein arginine methyltransferase.
    J Biol Chem. 1999 Jan 8;274(2):814-24 PMID: 9873020
  64. The yeast Saccharomyces cerevisiae YDL112w ORF encodes the putative 2'-O-ribose methyltransferase catalyzing the formation of Gm18 in tRNAs.
    RNA. 1999 Jan;5(1):66-81 PMID: 9917067
  65. GCD14p, a repressor of GCN4 translation, cooperates with Gcd10p and Lhp1p in the maturation of initiator methionyl-tRNA in Saccharomyces cerevisiae.
    Mol Cell Biol. 1999 Jun;19(6):4167-81 PMID: 10330157
Article Info
Journal
RNA (New York, N.Y.)
Abbr.
RNA
ISSN
1355-8382
DOI
10.1017/s1355838200992422
Published
2000-06-00
Pages
844-60
Language
English
Region
United States
NLM ID
9509184
PMCID
PMC1369962
Subset
IM
Databases
GENBANK
AJ250970
External Links
PubMed source DOI: 10.1017/s1355838200992422 PMC Full Text View journal details
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