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. 2002 Feb;8(2):202–213. doi: 10.1017/s1355838202013365

Posttranscriptional modifications in the A-loop of 23S rRNAs from selected archaea and eubacteria.

M A Hansen 1, F Kirpekar 1, W Ritterbusch 1, B Vester 1
PMCID: PMC1370243  PMID: 11911366

Abstract

Posttranscriptional modifications were mapped in helices 90-92 of 23S rRNA from the following phylogenetically diverse organisms: Haloarcula marismortui, Sulfolobus acidocaldarius, Bacillus subtilis, and Bacillus stearothermophilus. Helix 92 is a component of the ribosomal A-site, which contacts the aminoacyl-tRNA during protein synthesis, implying that posttranscriptional modifications in helices 90-92 may be important for ribosome function. RNA fragments were isolated from 23S rRNA by site-directed RNase H digestion. A novel method of mapping modifications by analysis of short, nucleotide-specific, RNase digestion fragments with Matrix Assisted Laser Desorption/Ionization Mass Spectrometry (MALDI-MS) was utilized. The MALDI-MS data were complemented by two primer extension techniques using reverse transcriptase. One technique utilizes decreasing concentrations of deoxynucleotide triphosphates to map 2'-O-ribose methylations. In the other, the rRNA is chemically modified, followed by mild alkaline hydrolysis to map pseudouridines (psis). A total of 10 posttranscriptionally methylated nucleotides and 6 psis were detected in the five organisms. Eight of the methylated nucleotides and one psi have not been reported previously. The distribution of modified nucleotides and their locations on the surface of the ribosomal peptidyl transferase cleft suggests functional importance.

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Selected References

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

  1. Baer R. J., Dubin D. T. Methylated regions of hamster mitochondrial ribosomal RNA: structural and functional correlates. Nucleic Acids Res. 1981 Jan 24;9(2):323–337. doi: 10.1093/nar/9.2.323. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. 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]
  3. Ban N., Nissen P., Hansen J., Moore P. B., Steitz T. A. The complete atomic structure of the large ribosomal subunit at 2.4 A resolution. Science. 2000 Aug 11;289(5481):905–920. doi: 10.1126/science.289.5481.905. [DOI] [PubMed] [Google Scholar]
  4. Blanchard S. C., Puglisi J. D. Solution structure of the A loop of 23S ribosomal RNA. Proc Natl Acad Sci U S A. 2001 Mar 20;98(7):3720–3725. doi: 10.1073/pnas.051608498. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bocchetta M., Xiong L., Mankin A. S. 23S rRNA positions essential for tRNA binding in ribosomal functional sites. Proc Natl Acad Sci U S A. 1998 Mar 31;95(7):3525–3530. doi: 10.1073/pnas.95.7.3525. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Branlant C., Krol A., Machatt M. A., Pouyet J., Ebel J. P., Edwards K., Kössel H. Primary and secondary structures of Escherichia coli MRE 600 23S ribosomal RNA. Comparison with models of secondary structure for maize chloroplast 23S rRNA and for large portions of mouse and human 16S mitochondrial rRNAs. Nucleic Acids Res. 1981 Sep 11;9(17):4303–4324. doi: 10.1093/nar/9.17.4303. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Brimacombe R., Mitchell P., Osswald M., Stade K., Bochkariov D. Clustering of modified nucleotides at the functional center of bacterial ribosomal RNA. FASEB J. 1993 Jan;7(1):161–167. doi: 10.1096/fasebj.7.1.8422963. [DOI] [PubMed] [Google Scholar]
  8. Brock T. D., Brock K. M., Belly R. T., Weiss R. L. Sulfolobus: a new genus of sulfur-oxidizing bacteria living at low pH and high temperature. Arch Mikrobiol. 1972;84(1):54–68. doi: 10.1007/BF00408082. [DOI] [PubMed] [Google Scholar]
  9. Caldas T., Binet E., Bouloc P., Richarme G. Translational defects of Escherichia coli mutants deficient in the Um(2552) 23S ribosomal RNA methyltransferase RrmJ/FTSJ. Biochem Biophys Res Commun. 2000 May 19;271(3):714–718. doi: 10.1006/bbrc.2000.2702. [DOI] [PubMed] [Google Scholar]
  10. Eladari M. E., Hampe A., Galibert F. Nucleotide sequence neighbouring a late modified guanylic residue within the 28S ribosomal RNA of several eukaryotic cells. Nucleic Acids Res. 1977 Jun;4(6):1759–1767. doi: 10.1093/nar/4.6.1759. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gaspin C., Cavaillé J., Erauso G., Bachellerie J. P. Archaeal homologs of eukaryotic methylation guide small nucleolar RNAs: lessons from the Pyrococcus genomes. J Mol Biol. 2000 Apr 7;297(4):895–906. doi: 10.1006/jmbi.2000.3593. [DOI] [PubMed] [Google Scholar]
  12. Green R., Noller H. F. In vitro complementation analysis localizes 23S rRNA posttranscriptional modifications that are required for Escherichia coli 50S ribosomal subunit assembly and function. RNA. 1996 Oct;2(10):1011–1021. [PMC free article] [PubMed] [Google Scholar]
  13. Green R., Noller H. F. Reconstitution of functional 50S ribosomes from in vitro transcripts of Bacillus stearothermophilus 23S rRNA. Biochemistry. 1999 Feb 9;38(6):1772–1779. doi: 10.1021/bi982246a. [DOI] [PubMed] [Google Scholar]
  14. Green R., Switzer C., Noller H. F. Ribosome-catalyzed peptide-bond formation with an A-site substrate covalently linked to 23S ribosomal RNA. Science. 1998 Apr 10;280(5361):286–289. doi: 10.1126/science.280.5361.286. [DOI] [PubMed] [Google Scholar]
  15. Humphrey W., Dalke A., Schulten K. VMD: visual molecular dynamics. J Mol Graph. 1996 Feb;14(1):33-8, 27-8. doi: 10.1016/0263-7855(96)00018-5. [DOI] [PubMed] [Google Scholar]
  16. Kawai G., Yamamoto Y., Kamimura T., Masegi T., Sekine M., Hata T., Iimori T., Watanabe T., Miyazawa T., Yokoyama S. Conformational rigidity of specific pyrimidine residues in tRNA arises from posttranscriptional modifications that enhance steric interaction between the base and the 2'-hydroxyl group. Biochemistry. 1992 Feb 4;31(4):1040–1046. doi: 10.1021/bi00119a012. [DOI] [PubMed] [Google Scholar]
  17. Khaitovich P., Tenson T., Kloss P., Mankin A. S. Reconstitution of functionally active Thermus aquaticus large ribosomal subunits with in vitro-transcribed rRNA. Biochemistry. 1999 Feb 9;38(6):1780–1788. doi: 10.1021/bi9822473. [DOI] [PubMed] [Google Scholar]
  18. Kim D. F., Green R. Base-pairing between 23S rRNA and tRNA in the ribosomal A site. Mol Cell. 1999 Nov;4(5):859–864. doi: 10.1016/s1097-2765(00)80395-0. [DOI] [PubMed] [Google Scholar]
  19. Kirpekar F., Douthwaite S., Roepstorff P. Mapping posttranscriptional modifications in 5S ribosomal RNA by MALDI mass spectrometry. RNA. 2000 Feb;6(2):296–306. doi: 10.1017/s1355838200992148. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kowalak J. A., Bruenger E., Hashizume T., Peltier J. M., Ofengand J., McCloskey J. A. Structural characterization of U*-1915 in domain IV from Escherichia coli 23S ribosomal RNA as 3-methylpseudouridine. Nucleic Acids Res. 1996 Feb 15;24(4):688–693. doi: 10.1093/nar/24.4.688. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kowalak J. A., Bruenger E., McCloskey J. A. Posttranscriptional modification of the central loop of domain V in Escherichia coli 23 S ribosomal RNA. J Biol Chem. 1995 Jul 28;270(30):17758–17764. doi: 10.1074/jbc.270.30.17758. [DOI] [PubMed] [Google Scholar]
  22. Lankat-Buttgereit B., Gross H. J., Krupp G. Detection of modified nucleosides by rapid RNA sequencing methods. Nucleic Acids Res. 1987 Sep 25;15(18):7649–7649. doi: 10.1093/nar/15.18.7649. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Lázaro E., Rodriguez-Fonseca C., Porse B., Ureña D., Garrett R. A., Ballesta J. P. A sparsomycin-resistant mutant of Halobacterium salinarium lacks a modification at nucleotide U2603 in the peptidyl transferase centre of 23 S rRNA. J Mol Biol. 1996 Aug 16;261(2):231–238. doi: 10.1006/jmbi.1996.0455. [DOI] [PubMed] [Google Scholar]
  24. Maden B. E., Corbett M. E., Heeney P. A., Pugh K., Ajuh P. M. Classical and novel approaches to the detection and localization of the numerous modified nucleotides in eukaryotic ribosomal RNA. Biochimie. 1995;77(1-2):22–29. doi: 10.1016/0300-9084(96)88100-4. [DOI] [PubMed] [Google Scholar]
  25. Maden B. E. Locations of methyl groups in 28 S rRNA of Xenopus laevis and man. Clustering in the conserved core of molecule. J Mol Biol. 1988 May 20;201(2):289–314. doi: 10.1016/0022-2836(88)90139-8. [DOI] [PubMed] [Google Scholar]
  26. 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]
  27. 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]
  28. Maxwell E. S., Fournier M. J. The small nucleolar RNAs. Annu Rev Biochem. 1995;64:897–934. doi: 10.1146/annurev.bi.64.070195.004341. [DOI] [PubMed] [Google Scholar]
  29. Moazed D., Noller H. F. Interaction of tRNA with 23S rRNA in the ribosomal A, P, and E sites. Cell. 1989 May 19;57(4):585–597. doi: 10.1016/0092-8674(89)90128-1. [DOI] [PubMed] [Google Scholar]
  30. 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]
  31. Nordhoff E., Cramer R., Karas M., Hillenkamp F., Kirpekar F., Kristiansen K., Roepstorff P. Ion stability of nucleic acids in infrared matrix-assisted laser desorption/ionization mass spectrometry. Nucleic Acids Res. 1993 Jul 25;21(15):3347–3357. doi: 10.1093/nar/21.15.3347. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. O'Connor M., Dahlberg A. E. Mutations at U2555, a tRNA-protected base in 23S rRNA, affect translational fidelity. Proc Natl Acad Sci U S A. 1993 Oct 1;90(19):9214–9218. doi: 10.1073/pnas.90.19.9214. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. 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]
  34. Ofengand J., Bakin A., Wrzesinski J., Nurse K., Lane B. G. The pseudouridine residues of ribosomal RNA. Biochem Cell Biol. 1995 Nov-Dec;73(11-12):915–924. doi: 10.1139/o95-099. [DOI] [PubMed] [Google Scholar]
  35. Omer A. D., Lowe T. M., Russell A. G., Ebhardt H., Eddy S. R., Dennis P. P. Homologs of small nucleolar RNAs in Archaea. Science. 2000 Apr 21;288(5465):517–522. doi: 10.1126/science.288.5465.517. [DOI] [PubMed] [Google Scholar]
  36. Ostergaard P., Phan H., Johansen L. B., Egebjerg J., Ostergaard L., Porse B. T., Garrett R. A. Assembly of proteins and 5 S rRNA to transcripts of the major structural domains of 23 S rRNA. J Mol Biol. 1998 Nov 27;284(2):227–240. doi: 10.1006/jmbi.1998.2185. [DOI] [PubMed] [Google Scholar]
  37. Porse B. T., Garrett R. A. Mapping important nucleotides in the peptidyl transferase centre of 23 S rRNA using a random mutagenesis approach. J Mol Biol. 1995 May 26;249(1):1–10. doi: 10.1006/jmbi.1995.0276. [DOI] [PubMed] [Google Scholar]
  38. Rodriguez-Fonseca C., Phan H., Long K. S., Porse B. T., Kirillov S. V., Amils R., Garrett R. A. Puromycin-rRNA interaction sites at the peptidyl transferase center. RNA. 2000 May;6(5):744–754. doi: 10.1017/s1355838200000091. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Rozenski J., Crain P. F., McCloskey J. A. The RNA Modification Database: 1999 update. Nucleic Acids Res. 1999 Jan 1;27(1):196–197. doi: 10.1093/nar/27.1.196. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Sirum-Connolly K., Peltier J. M., Crain P. F., McCloskey J. A., Mason T. L. Implications of a functional large ribosomal RNA with only three modified nucleotides. Biochimie. 1995;77(1-2):30–39. doi: 10.1016/0300-9084(96)88101-6. [DOI] [PubMed] [Google Scholar]
  41. Smith C. M., Steitz J. A. Sno storm in the nucleolus: new roles for myriad small RNPs. Cell. 1997 May 30;89(5):669–672. doi: 10.1016/s0092-8674(00)80247-0. [DOI] [PubMed] [Google Scholar]
  42. Smith J. E., Cooperman B. S., Mitchell P. Methylation sites in Escherichia coli ribosomal RNA: localization and identification of four new sites of methylation in 23S rRNA. Biochemistry. 1992 Nov 10;31(44):10825–10834. doi: 10.1021/bi00159a025. [DOI] [PubMed] [Google Scholar]
  43. Stern S., Moazed D., Noller H. F. Structural analysis of RNA using chemical and enzymatic probing monitored by primer extension. Methods Enzymol. 1988;164:481–489. doi: 10.1016/s0076-6879(88)64064-x. [DOI] [PubMed] [Google Scholar]
  44. Veldman G. M., Klootwijk J., de Regt V. C., Planta R. J., Branlant C., Krol A., Ebel J. P. The primary and secondary structure of yeast 26S rRNA. Nucleic Acids Res. 1981 Dec 21;9(24):6935–6952. doi: 10.1093/nar/9.24.6935. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Vester B., Douthwaite S. Domain V of 23S rRNA contains all the structural elements necessary for recognition by the ErmE methyltransferase. J Bacteriol. 1994 Nov;176(22):6999–7004. doi: 10.1128/jb.176.22.6999-7004.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Vester B., Nielsen A. K., Hansen L. H., Douthwaite S. ErmE methyltransferase recognition elements in RNA substrates. J Mol Biol. 1998 Sep 18;282(2):255–264. doi: 10.1006/jmbi.1998.2024. [DOI] [PubMed] [Google Scholar]
  47. Welch M., Chastang J., Yarus M. An inhibitor of ribosomal peptidyl transferase using transition-state analogy. Biochemistry. 1995 Jan 17;34(2):385–390. doi: 10.1021/bi00002a001. [DOI] [PubMed] [Google Scholar]
  48. Yusupov M. M., Yusupova G. Z., Baucom A., Lieberman K., Earnest T. N., Cate J. H., Noller H. F. Crystal structure of the ribosome at 5.5 A resolution. Science. 2001 Mar 29;292(5518):883–896. doi: 10.1126/science.1060089. [DOI] [PubMed] [Google Scholar]

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