Skip to main content
NCBI home page
RNA logoLink to RNA
. 2000 Jun;6(6):844–860. doi: 10.1017/s1355838200992422

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

M E Nordlund 1, J O Johansson 1, U von Pawel-Rammingen 1, A S Byström 1
PMCID: PMC1369962  PMID: 10864043

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.

Full Text

The Full Text of this article is available as a PDF (599.2 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Anderson J., Phan L., Cuesta R., Carlson B. A., Pak M., Asano K., Björk G. R., Tamame M., Hinnebusch A. G. 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):3650–3662. doi: 10.1101/gad.12.23.3650. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Asefa B., Kauler P., Cournoyer D., Lehnert S., Chow T. Y. Genetic analysis of the yeast NUD1 endo-exonuclease: a role in the repair of DNA double-strand breaks. Curr Genet. 1998 Dec;34(5):360–367. doi: 10.1007/s002940050407. [DOI] [PubMed] [Google Scholar]
  3. Aström S. U., Byström A. S. Rit1, a tRNA backbone-modifying enzyme that mediates initiator and elongator tRNA discrimination. Cell. 1994 Nov 4;79(3):535–546. doi: 10.1016/0092-8674(94)90262-3. [DOI] [PubMed] [Google Scholar]
  4. Aström S. U., von Pawel-Rammingen U., Byström A. S. The yeast initiator tRNAMet can act as an elongator tRNA(Met) in vivo. J Mol Biol. 1993 Sep 5;233(1):43–58. doi: 10.1006/jmbi.1993.1483. [DOI] [PubMed] [Google Scholar]
  5. Avital S., Elson D. A convenient procedure for preparing transfer ribonucleic acid from Escherichia coli. Biochim Biophys Acta. 1969 Apr 22;179(2):297–307. doi: 10.1016/0005-2787(69)90038-0. [DOI] [PubMed] [Google Scholar]
  6. Bai Y., Symington L. S. A Rad52 homolog is required for RAD51-independent mitotic recombination in Saccharomyces cerevisiae. Genes Dev. 1996 Aug 15;10(16):2025–2037. doi: 10.1101/gad.10.16.2025. [DOI] [PubMed] [Google Scholar]
  7. Becker H. F., Motorin Y., Planta R. J., Grosjean H. 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):4493–4499. doi: 10.1093/nar/25.22.4493. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Bellisario R. L., Maley G. F., Galivan J. H., Maley F. Amino acid sequence at the FdUMP binding site of thymidylate synthetase. Proc Natl Acad Sci U S A. 1976 Jun;73(6):1848–1852. doi: 10.1073/pnas.73.6.1848. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Björk G. R., Neidhardt F. C. 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. doi: 10.1128/jb.124.1.99-111.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Boeke J. D., Garfinkel D. J., Styles C. A., Fink G. R. Ty elements transpose through an RNA intermediate. Cell. 1985 Mar;40(3):491–500. doi: 10.1016/0092-8674(85)90197-7. [DOI] [PubMed] [Google Scholar]
  11. Buck M., Connick M., Ames B. N. 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. doi: 10.1016/0003-2697(83)90044-1. [DOI] [PubMed] [Google Scholar]
  12. Byström A. S., Fink G. R. A functional analysis of the repeated methionine initiator tRNA genes (IMT) in yeast. Mol Gen Genet. 1989 Apr;216(2-3):276–286. doi: 10.1007/BF00334366. [DOI] [PubMed] [Google Scholar]
  13. Calvo O., Cuesta R., Anderson J., Gutiérrez N., García-Barrio M. T., Hinnebusch A. G., Tamame M. 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):4167–4181. doi: 10.1128/mcb.19.6.4167. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Carlson M., Botstein D. Two differentially regulated mRNAs with different 5' ends encode secreted with intracellular forms of yeast invertase. Cell. 1982 Jan;28(1):145–154. doi: 10.1016/0092-8674(82)90384-1. [DOI] [PubMed] [Google Scholar]
  15. Cavaillé J., Chetouani F., Bachellerie J. P. 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. doi: 10.1017/s1355838299981475. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Chow T. Y., Fraser M. J. Purification and properties of single strand DNA-binding endo-exonuclease of Neurospora crassa. J Biol Chem. 1983 Oct 10;258(19):12010–12018. [PubMed] [Google Scholar]
  17. Chow T. Y., Kunz B. A. 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. doi: 10.1007/BF00312763. [DOI] [PubMed] [Google Scholar]
  18. Chow T. Y., Perkins E. L., Resnick M. A. Yeast RNC1 encodes a chimeric protein, RhoNUC, with a human rho motif and deoxyribonuclease activity. Nucleic Acids Res. 1992 Oct 11;20(19):5215–5221. doi: 10.1093/nar/20.19.5215. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Chow T. Y., Resnick M. A. An endo-exonuclease activity of yeast that requires a functional RAD52 gene. Mol Gen Genet. 1988 Jan;211(1):41–48. doi: 10.1007/BF00338391. [DOI] [PubMed] [Google Scholar]
  20. Chow T. Y., Resnick M. A. 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):17659–17667. [PubMed] [Google Scholar]
  21. Chu S., DeRisi J., Eisen M., Mulholland J., Botstein D., Brown P. O., Herskowitz I. The transcriptional program of sporulation in budding yeast. Science. 1998 Oct 23;282(5389):699–705. doi: 10.1126/science.282.5389.699. [DOI] [PubMed] [Google Scholar]
  22. Curcio M. J., Garfinkel D. J. Heterogeneous functional Ty1 elements are abundant in the Saccharomyces cerevisiae genome. Genetics. 1994 Apr;136(4):1245–1259. doi: 10.1093/genetics/136.4.1245. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Dihanich M. E., Najarian D., Clark R., Gillman E. C., Martin N. C., Hopper A. K. 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):177–184. doi: 10.1128/mcb.7.1.177. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Ellis S. R., Morales M. J., Li J. M., Hopper A. K., Martin N. C. 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):9703–9709. [PubMed] [Google Scholar]
  25. Fiorentini P., Huang K. N., Tishkoff D. X., Kolodner R. D., Symington L. S. Exonuclease I of Saccharomyces cerevisiae functions in mitotic recombination in vivo and in vitro. Mol Cell Biol. 1997 May;17(5):2764–2773. doi: 10.1128/mcb.17.5.2764. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Fraser M. J., Koa H., Chow T. Y. Neurospora endo-exonuclease is immunochemically related to the recC gene product of Escherichia coli. J Bacteriol. 1990 Jan;172(1):507–510. doi: 10.1128/jb.172.1.507-510.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Gehrke C. W., Kuo K. C., McCune R. A., Gerhardt K. O., Agris P. F. Quantitative enzymatic hydrolysis of tRNAs: reversed-phase high-performance liquid chromatography of tRNA nucleosides. J Chromatogr. 1982 Jul 9;230(2):297–308. [PubMed] [Google Scholar]
  28. Gerber A. P., Keller W. An adenosine deaminase that generates inosine at the wobble position of tRNAs. Science. 1999 Nov 5;286(5442):1146–1149. doi: 10.1126/science.286.5442.1146. [DOI] [PubMed] [Google Scholar]
  29. Gietz D., St Jean A., Woods R. A., Schiestl R. H. Improved method for high efficiency transformation of intact yeast cells. Nucleic Acids Res. 1992 Mar 25;20(6):1425–1425. doi: 10.1093/nar/20.6.1425. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Gu X. R., Santi D. V. High-level expression of Escherichia coli tRNA (m5U54)-methyltransferase. DNA Cell Biol. 1990 May;9(4):273–278. doi: 10.1089/dna.1990.9.273. [DOI] [PubMed] [Google Scholar]
  31. Gustafsson C., Lindström P. H., Hagervall T. G., Esberg K. B., Björk G. R. 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):1757–1764. doi: 10.1128/jb.173.5.1757-1764.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Göransson M., Forsman P., Nilsson P., Uhlin B. E. 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):1557–1565. doi: 10.1111/j.1365-2958.1989.tb00141.x. [DOI] [PubMed] [Google Scholar]
  33. Herskowitz I., Jensen R. E. Putting the HO gene to work: practical uses for mating-type switching. Methods Enzymol. 1991;194:132–146. doi: 10.1016/0076-6879(91)94011-z. [DOI] [PubMed] [Google Scholar]
  34. Hill J. E., Myers A. M., Koerner T. J., Tzagoloff A. Yeast/E. coli shuttle vectors with multiple unique restriction sites. Yeast. 1986 Sep;2(3):163–167. doi: 10.1002/yea.320020304. [DOI] [PubMed] [Google Scholar]
  35. Hopper A. K., Furukawa A. H., Pham H. D., Martin N. C. Defects in modification of cytoplasmic and mitochondrial transfer RNAs are caused by single nuclear mutations. Cell. 1982 Mar;28(3):543–550. doi: 10.1016/0092-8674(82)90209-4. [DOI] [PubMed] [Google Scholar]
  36. Kagan R. M., Clarke S. 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):417–427. doi: 10.1006/abbi.1994.1187. [DOI] [PubMed] [Google Scholar]
  37. Kealey J. T., Gu X., Santi D. V. Enzymatic mechanism of tRNA (m5U54)methyltransferase. Biochimie. 1994;76(12):1133–1142. doi: 10.1016/0300-9084(94)90042-6. [DOI] [PubMed] [Google Scholar]
  38. Kealey J. T., Santi D. V. Identification of the catalytic nucleophile of tRNA (m5U54)methyltransferase. Biochemistry. 1991 Oct 8;30(40):9724–9728. doi: 10.1021/bi00104a022. [DOI] [PubMed] [Google Scholar]
  39. Kersten H., Albani M., Männlein E., Praisler R., Wurmbach P., Nierhaus K. H. On the role of ribosylthymine in prokaryotic tRNA function. Eur J Biochem. 1981 Feb;114(2):451–456. doi: 10.1111/j.1432-1033.1981.tb05166.x. [DOI] [PubMed] [Google Scholar]
  40. Kozak M. The scanning model for translation: an update. J Cell Biol. 1989 Feb;108(2):229–241. doi: 10.1083/jcb.108.2.229. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Kunkel T. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A. 1985 Jan;82(2):488–492. doi: 10.1073/pnas.82.2.488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Lecointe F., Simos G., Sauer A., Hurt E. C., Motorin Y., Grosjean H. 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):1316–1323. doi: 10.1074/jbc.273.3.1316. [DOI] [PubMed] [Google Scholar]
  43. Makkerh J. P., Dingwall C., Laskey R. A. Comparative mutagenesis of nuclear localization signals reveals the importance of neutral and acidic amino acids. Curr Biol. 1996 Aug 1;6(8):1025–1027. doi: 10.1016/s0960-9822(02)00648-6. [DOI] [PubMed] [Google Scholar]
  44. Malone R. E., Esposito R. E. 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):503–507. doi: 10.1073/pnas.77.1.503. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Marsischky G. T., Filosi N., Kane M. F., Kolodner R. Redundancy of Saccharomyces cerevisiae MSH3 and MSH6 in MSH2-dependent mismatch repair. Genes Dev. 1996 Feb 15;10(4):407–420. doi: 10.1101/gad.10.4.407. [DOI] [PubMed] [Google Scholar]
  46. Motorin Y., Grosjean H. 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):1105–1118. doi: 10.1017/s1355838299982201. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Myers A. M., Tzagoloff A., Kinney D. M., Lusty C. J. Yeast shuttle and integrative vectors with multiple cloning sites suitable for construction of lacZ fusions. Gene. 1986;45(3):299–310. doi: 10.1016/0378-1119(86)90028-4. [DOI] [PubMed] [Google Scholar]
  48. Niewmierzycka A., Clarke S. S-Adenosylmethionine-dependent methylation in Saccharomyces cerevisiae. Identification of a novel protein arginine methyltransferase. J Biol Chem. 1999 Jan 8;274(2):814–824. doi: 10.1074/jbc.274.2.814. [DOI] [PubMed] [Google Scholar]
  49. Orr-Weaver T. L., Szostak J. W., Rothstein R. J. Yeast transformation: a model system for the study of recombination. Proc Natl Acad Sci U S A. 1981 Oct;78(10):6354–6358. doi: 10.1073/pnas.78.10.6354. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Osterman D. G., DePillis G. D., Wu J. C., Matsuda A., Santi D. V. 5-Fluorocytosine in DNA is a mechanism-based inhibitor of HhaI methylase. Biochemistry. 1988 Jul 12;27(14):5204–5210. doi: 10.1021/bi00414a039. [DOI] [PubMed] [Google Scholar]
  51. Persson B. C., Gustafsson C., Berg D. E., Björk G. R. 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):3995–3998. doi: 10.1073/pnas.89.9.3995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Pomerantz S. C., McCloskey J. A. Analysis of RNA hydrolyzates by liquid chromatography-mass spectrometry. Methods Enzymol. 1990;193:796–824. doi: 10.1016/0076-6879(90)93452-q. [DOI] [PubMed] [Google Scholar]
  53. Resnick M. A., Sugino A., Nitiss J., Chow T. DNA polymerases, deoxyribonucleases, and recombination during meiosis in Saccharomyces cerevisiae. Mol Cell Biol. 1984 Dec;4(12):2811–2817. doi: 10.1128/mcb.4.12.2811. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Rose A. M., Joyce P. B., Hopper A. K., Martin N. C. 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):5652–5658. doi: 10.1128/mcb.12.12.5652. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Scherer S., Davis R. W. Replacement of chromosome segments with altered DNA sequences constructed in vitro. Proc Natl Acad Sci U S A. 1979 Oct;76(10):4951–4955. doi: 10.1073/pnas.76.10.4951. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Sikorski R. S., Hieter P. 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. doi: 10.1093/genetics/122.1.19. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Simos G., Tekotte H., Grosjean H., Segref A., Sharma K., Tollervey D., Hurt E. C. Nuclear pore proteins are involved in the biogenesis of functional tRNA. EMBO J. 1996 May 1;15(9):2270–2284. [PMC free article] [PubMed] [Google Scholar]
  59. Smith D. B., Johnson K. S. Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene. 1988 Jul 15;67(1):31–40. doi: 10.1016/0378-1119(88)90005-4. [DOI] [PubMed] [Google Scholar]
  60. Studier F. W., Moffatt B. A. Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol. 1986 May 5;189(1):113–130. doi: 10.1016/0022-2836(86)90385-2. [DOI] [PubMed] [Google Scholar]
  61. Tatusova T. A., Madden T. L. BLAST 2 Sequences, a new tool for comparing protein and nucleotide sequences. FEMS Microbiol Lett. 1999 May 15;174(2):247–250. doi: 10.1111/j.1574-6968.1999.tb13575.x. [DOI] [PubMed] [Google Scholar]
  62. Tolerico L. H., Benko A. L., Aris J. P., Stanford D. R., Martin N. C., Hopper A. K. Saccharomyces cerevisiae Mod5p-II contains sequences antagonistic for nuclear and cytosolic locations. Genetics. 1999 Jan;151(1):57–75. doi: 10.1093/genetics/151.1.57. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Van Vliet-Reedijk J. C., Planta R. J. The RHO4a and NUD1 genes on Saccharomyces cerevisiae chromosome XI. Yeast. 1993 Oct;9(10):1139–1147. doi: 10.1002/yea.320091015. [DOI] [PubMed] [Google Scholar]
  64. Wu J. C., Santi D. V. Kinetic and catalytic mechanism of HhaI methyltransferase. J Biol Chem. 1987 Apr 5;262(10):4778–4786. [PubMed] [Google Scholar]
  65. von Pawel-Rammingen U., Aström S., Byström A. S. Mutational analysis of conserved positions potentially important for initiator tRNA function in Saccharomyces cerevisiae. Mol Cell Biol. 1992 Apr;12(4):1432–1442. doi: 10.1128/mcb.12.4.1432. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from RNA are provided here courtesy of The RNA Society

RESOURCES