Skip to main content
RNA logoLink to RNA
. 2001 Nov;7(11):1603–1615.

Identification and site of action of the remaining four putative pseudouridine synthases in Escherichia coli.

M Del Campo 1, Y Kaya 1, J Ofengand 1
PMCID: PMC1370202  PMID: 11720289

Abstract

There are 10 known putative pseudouridine synthase genes in Escherichia coli. The products of six have been previously assigned, one to formation of the single pseudouridine in 16S RNA, three to the formation of seven pseudouridines in 23S RNA, and three to the formation of three pseudouridines in tRNA (one synthase makes pseudouridine in 23S RNA and tRNA). Here we show that the remaining four putative synthase genes make bona fide pseudouridine synthases and identify which pseudouridines they make. RluB (formerly YciL) and RluE (formerly YmfC) make pseudouridine2605 and pseudouridine2457, respectively, in 23S RNA. RluF (formerly YjbC) makes the newly discovered pseudouridine2604 in 23S RNA, and TruC (formerly YqcB) makes pseudouridine65 in tRNA(Ile1) and tRNA(Asp). Deletion of each of these synthase genes individually had no effect on exponential growth in rich media at 25 degrees C, 37 degrees C, or 42 degrees C. A strain lacking RluB and RluF also showed no growth defect under these conditions. Mutation of a conserved aspartate in a common sequence motif, previously shown to be essential for the other six E. coli pseudouridine synthases and several yeast pseudouridine synthases, also caused a loss of in vivo activity in all four of the synthases studied in this work.

Full Text

The Full Text of this article is available as a PDF (2.0 MB).

Selected References

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

  1. Ansmant I., Massenet S., Grosjean H., Motorin Y., Branlant C. Identification of the Saccharomyces cerevisiae RNA:pseudouridine synthase responsible for formation of psi(2819) in 21S mitochondrial ribosomal RNA. Nucleic Acids Res. 2000 May 1;28(9):1941–1946. doi: 10.1093/nar/28.9.1941. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Ansmant I., Motorin Y., Massenet S., Grosjean H., Branlant C. Identification and characterization of the tRNA:Psi 31-synthase (Pus6p) of Saccharomyces cerevisiae. J Biol Chem. 2001 Jun 13;276(37):34934–34940. doi: 10.1074/jbc.M103131200. [DOI] [PubMed] [Google Scholar]
  3. Arps P. J., Marvel C. C., Rubin B. C., Tolan D. A., Penhoet E. E., Winkler M. E. Structural features of the hisT operon of Escherichia coli K-12. Nucleic Acids Res. 1985 Jul 25;13(14):5297–5315. doi: 10.1093/nar/13.14.5297. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bakin A. V., Ofengand J. Mapping of pseudouridine residues in RNA to nucleotide resolution. Methods Mol Biol. 1998;77:297–309. doi: 10.1385/0-89603-397-X:297. [DOI] [PubMed] [Google Scholar]
  5. Bakin A., Kowalak J. A., McCloskey J. A., Ofengand J. The single pseudouridine residue in Escherichia coli 16S RNA is located at position 516. Nucleic Acids Res. 1994 Sep 11;22(18):3681–3684. doi: 10.1093/nar/22.18.3681. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bakin A., Lane B. G., Ofengand J. Clustering of pseudouridine residues around the peptidyltransferase center of yeast cytoplasmic and mitochondrial ribosomes. Biochemistry. 1994 Nov 15;33(45):13475–13483. doi: 10.1021/bi00249a036. [DOI] [PubMed] [Google Scholar]
  7. Bakin A., Ofengand J. Four newly located pseudouridylate residues in Escherichia coli 23S ribosomal RNA are all at the peptidyltransferase center: analysis by the application of a new sequencing technique. Biochemistry. 1993 Sep 21;32(37):9754–9762. doi: 10.1021/bi00088a030. [DOI] [PubMed] [Google Scholar]
  8. Ciampi M. S., Arena F., Cortese R. Biosynthesis of pseudouridine in the in vitro transcribed tRNATyr precursor. FEBS Lett. 1977 May 1;77(1):75–82. doi: 10.1016/0014-5793(77)80196-8. [DOI] [PubMed] [Google Scholar]
  9. Colosimo A., Xu Z., Novelli G., Dallapiccola B., Gruenert D. C. Simple version of "megaprimer" PCR for site-directed mutagenesis. Biotechniques. 1999 May;26(5):870–873. doi: 10.2144/99265bm15. [DOI] [PubMed] [Google Scholar]
  10. Conrad J., Niu L., Rudd K., Lane B. G., Ofengand J. 16S ribosomal RNA pseudouridine synthase RsuA of Escherichia coli: deletion, mutation of the conserved Asp102 residue, and sequence comparison among all other pseudouridine synthases. RNA. 1999 Jun;5(6):751–763. doi: 10.1017/s1355838299990167. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Conrad J., Sun D., Englund N., Ofengand J. The rluC gene of Escherichia coli codes for a pseudouridine synthase that is solely responsible for synthesis of pseudouridine at positions 955, 2504, and 2580 in 23 S ribosomal RNA. J Biol Chem. 1998 Jul 17;273(29):18562–18566. doi: 10.1074/jbc.273.29.18562. [DOI] [PubMed] [Google Scholar]
  12. Deutscher M. P., Hilderman R. H. Isolation and partial characterization of Escherichia coli mutants with low levels of transfer ribonucleic acid nucleotidyltransferase. J Bacteriol. 1974 May;118(2):621–627. doi: 10.1128/jb.118.2.621-627.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gustafsson C., Reid R., Greene P. J., Santi D. V. Identification of new RNA modifying enzymes by iterative genome search using known modifying enzymes as probes. Nucleic Acids Res. 1996 Oct 1;24(19):3756–3762. doi: 10.1093/nar/24.19.3756. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Gutell R. R., Gray M. W., Schnare M. N. A compilation of large subunit (23S and 23S-like) ribosomal RNA structures: 1993. Nucleic Acids Res. 1993 Jul 1;21(13):3055–3074. doi: 10.1093/nar/21.13.3055. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Gutgsell N. S., Del Campo M., Raychaudhuri S., Ofengand J. A second function for pseudouridine synthases: A point mutant of RluD unable to form pseudouridines 1911, 1915, and 1917 in Escherichia coli 23S ribosomal RNA restores normal growth to an RluD-minus strain. RNA. 2001 Jul;7(7):990–998. doi: 10.1017/s1355838201000243. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Gutgsell N., Englund N., Niu L., Kaya Y., Lane B. G., Ofengand J. Deletion of the Escherichia coli pseudouridine synthase gene truB blocks formation of pseudouridine 55 in tRNA in vivo, does not affect exponential growth, but confers a strong selective disadvantage in competition with wild-type cells. RNA. 2000 Dec;6(12):1870–1881. doi: 10.1017/s1355838200001588. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Huang L., Ku J., Pookanjanatavip M., Gu X., Wang D., Greene P. J., Santi D. V. Identification of two Escherichia coli pseudouridine synthases that show multisite specificity for 23S RNA. Biochemistry. 1998 Nov 10;37(45):15951–15957. doi: 10.1021/bi981002n. [DOI] [PubMed] [Google Scholar]
  18. Huang L., Pookanjanatavip M., Gu X., Santi D. V. A conserved aspartate of tRNA pseudouridine synthase is essential for activity and a probable nucleophilic catalyst. Biochemistry. 1998 Jan 6;37(1):344–351. doi: 10.1021/bi971874+. [DOI] [PubMed] [Google Scholar]
  19. Johnson L., Söll D. In vitro biosynthesis of pseudouridine at the polynucleotide level by an enzyme extract from Escherichia coli. Proc Natl Acad Sci U S A. 1970 Oct;67(2):943–950. doi: 10.1073/pnas.67.2.943. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kammen H. O., Marvel C. C., Hardy L., Penhoet E. E. Purification, structure, and properties of Escherichia coli tRNA pseudouridine synthase I. J Biol Chem. 1988 Feb 15;263(5):2255–2263. [PubMed] [Google Scholar]
  21. Koonin E. V. Pseudouridine synthases: four families of enzymes containing a putative uridine-binding motif also conserved in dUTPases and dCTP deaminases. Nucleic Acids Res. 1996 Jun 15;24(12):2411–2415. doi: 10.1093/nar/24.12.2411. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Link A. J., Phillips D., Church G. M. Methods for generating precise deletions and insertions in the genome of wild-type Escherichia coli: application to open reading frame characterization. J Bacteriol. 1997 Oct;179(20):6228–6237. doi: 10.1128/jb.179.20.6228-6237.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Maden B. E. The numerous modified nucleotides in eukaryotic ribosomal RNA. Prog Nucleic Acid Res Mol Biol. 1990;39:241–303. doi: 10.1016/s0079-6603(08)60629-7. [DOI] [PubMed] [Google Scholar]
  24. Marvel C. C., Arps P. J., Rubin B. C., Kammen H. O., Penhoet E. E., Winkler M. E. hisT is part of a multigene operon in Escherichia coli K-12. J Bacteriol. 1985 Jan;161(1):60–71. doi: 10.1128/jb.161.1.60-71.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Massenet S., Ansmant I., Motorin Y., Branlant C. The first determination of pseudouridine residues in 23S ribosomal RNA from hyperthermophilic Archaea Sulfolobus acidocaldarius. FEBS Lett. 1999 Nov 26;462(1-2):94–100. doi: 10.1016/s0014-5793(99)01524-0. [DOI] [PubMed] [Google Scholar]
  26. Muth G. W., Ortoleva-Donnelly L., Strobel S. A. A single adenosine with a neutral pKa in the ribosomal peptidyl transferase center. Science. 2000 Aug 11;289(5481):947–950. doi: 10.1126/science.289.5481.947. [DOI] [PubMed] [Google Scholar]
  27. Nissen P., Hansen J., Ban N., Moore P. B., Steitz T. A. The structural basis of ribosome activity in peptide bond synthesis. Science. 2000 Aug 11;289(5481):920–930. doi: 10.1126/science.289.5481.920. [DOI] [PubMed] [Google Scholar]
  28. Niu L., Lane B. G., Ofengand J. Cloning and characterization of the 23S RNA pseudouridine 2633 synthase from Bacillus subtilis. Biochemistry. 1999 Jan 12;38(2):629–635. doi: 10.1021/bi9821869. [DOI] [PubMed] [Google Scholar]
  29. Nurse K., Wrzesinski J., Bakin A., Lane B. G., Ofengand J. Purification, cloning, and properties of the tRNA psi 55 synthase from Escherichia coli. RNA. 1995 Mar;1(1):102–112. [PMC free article] [PubMed] [Google Scholar]
  30. Ofengand J., Bakin A. Mapping to nucleotide resolution of pseudouridine residues in large subunit ribosomal RNAs from representative eukaryotes, prokaryotes, archaebacteria, mitochondria and chloroplasts. J Mol Biol. 1997 Feb 21;266(2):246–268. doi: 10.1006/jmbi.1996.0737. [DOI] [PubMed] [Google Scholar]
  31. Ramamurthy V., Swann S. L., Paulson J. L., Spedaliere C. J., Mueller E. G. Critical aspartic acid residues in pseudouridine synthases. J Biol Chem. 1999 Aug 6;274(32):22225–22230. doi: 10.1074/jbc.274.32.22225. [DOI] [PubMed] [Google Scholar]
  32. Raychaudhuri S., Conrad J., Hall B. G., Ofengand J. A pseudouridine synthase required for the formation of two universally conserved pseudouridines in ribosomal RNA is essential for normal growth of Escherichia coli. RNA. 1998 Nov;4(11):1407–1417. doi: 10.1017/s1355838298981146. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Raychaudhuri S., Niu L., Conrad J., Lane B. G., Ofengand J. Functional effect of deletion and mutation of the Escherichia coli ribosomal RNA and tRNA pseudouridine synthase RluA. J Biol Chem. 1999 Jul 2;274(27):18880–18886. doi: 10.1074/jbc.274.27.18880. [DOI] [PubMed] [Google Scholar]
  34. Samuelsson T., Olsson M. Transfer RNA pseudouridine synthases in Saccharomyces cerevisiae. J Biol Chem. 1990 May 25;265(15):8782–8787. [PubMed] [Google Scholar]
  35. 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]
  36. Wrzesinski J., Bakin A., Nurse K., Lane B. G., Ofengand J. Purification, cloning, and properties of the 16S RNA pseudouridine 516 synthase from Escherichia coli. Biochemistry. 1995 Jul 11;34(27):8904–8913. doi: 10.1021/bi00027a043. [DOI] [PubMed] [Google Scholar]
  37. Wrzesinski J., Nurse K., Bakin A., Lane B. G., Ofengand J. A dual-specificity pseudouridine synthase: an Escherichia coli synthase purified and cloned on the basis of its specificity for psi 746 in 23S RNA is also specific for psi 32 in tRNA(phe). RNA. 1995 Jun;1(4):437–448. [PMC free article] [PubMed] [Google Scholar]
  38. Zebarjadian Y., King T., Fournier M. J., Clarke L., Carbon J. Point mutations in yeast CBF5 can abolish in vivo pseudouridylation of rRNA. Mol Cell Biol. 1999 Nov;19(11):7461–7472. doi: 10.1128/mcb.19.11.7461. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from RNA are provided here courtesy of The RNA Society

RESOURCES