Skip to main content
PMC home page
The EMBO Journal logoLink to The EMBO Journal
. 1997 Oct 15;16(20):6290–6300. doi: 10.1093/emboj/16.20.6290

RNA-protein interactions of an archaeal homotetrameric splicing endoribonuclease with an exceptional evolutionary history.

J Lykke-Andersen 1, R A Garrett 1
PMCID: PMC1326313  PMID: 9321408

Abstract

The splicing endoribonuclease from Methanococcus jannaschii, a member of a recently defined family of enzymes involved in splicing of archaeal introns and eukaryotic nuclear tRNA introns, was isolated and shown by cross-linking studies to form a homotetramer in solution. A non-cleavable substrate analogue was synthesized by incorporating 2'-deoxyuridines at the two cleavage sites and complexed to the splicing enzyme. The complex was subjected to protein footprinting and the results implicated an RNP1-like sequence and a sequence region immediately N-terminal to a putative leucine zipper in substrate binding. In addition, a histidine residue (His125), positioned within a third RNA binding region, was shown to be involved in catalysis by mutagenesis. The splicing enzyme was localized on the central helix and the two 3 nt bulges of the conserved archaeal 'bulge-helix-bulge' substrate motif by RNA footprinting. Sequence comparison with the dimeric splicing enzyme from Halobacterium volcanii demonstrates that the latter is a tandemly repeated duplication of the former, where alternating segments within each protein half degenerated after the duplication event. Another duplication event, in the eukaryotic domain, produced two different homologues of the M.jannaschii-type enzyme structure. The data provide strong evidence that the tetrameric M.jannaschii enzyme consists of two isologously associated dimers, each similar to one H.volcanii monomer and each consisting of two monomers, where one face of monomer 1 and the opposite face of monomer 2 are involved in RNA binding.

Full Text

The Full Text of this article is available as a PDF (644.6 KB).

Selected References

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

  1. Belford H. G., Westaway S. K., Abelson J., Greer C. L. Multiple nucleotide cofactor use by yeast ligase in tRNA splicing. Evidence for independent ATP- and GTP-binding sites. J Biol Chem. 1993 Feb 5;268(4):2444–2450. [PubMed] [Google Scholar]
  2. Bult C. J., White O., Olsen G. J., Zhou L., Fleischmann R. D., Sutton G. G., Blake J. A., FitzGerald L. M., Clayton R. A., Gocayne J. D. Complete genome sequence of the methanogenic archaeon, Methanococcus jannaschii. Science. 1996 Aug 23;273(5278):1058–1073. doi: 10.1126/science.273.5278.1058. [DOI] [PubMed] [Google Scholar]
  3. Burggraf S., Larsen N., Woese C. R., Stetter K. O. An intron within the 16S ribosomal RNA gene of the archaeon Pyrobaculum aerophilum. Proc Natl Acad Sci U S A. 1993 Mar 15;90(6):2547–2550. doi: 10.1073/pnas.90.6.2547. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Cech T. R. Self-splicing of group I introns. Annu Rev Biochem. 1990;59:543–568. doi: 10.1146/annurev.bi.59.070190.002551. [DOI] [PubMed] [Google Scholar]
  5. Conrad F., Hanne A., Gaur R. K., Krupp G. Enzymatic synthesis of 2'-modified nucleic acids: identification of important phosphate and ribose moieties in RNase P substrates. Nucleic Acids Res. 1995 Jun 11;23(11):1845–1853. doi: 10.1093/nar/23.11.1845. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Dalgaard J. Z., Garrett R. A., Belfort M. A site-specific endonuclease encoded by a typical archaeal intron. Proc Natl Acad Sci U S A. 1993 Jun 15;90(12):5414–5417. doi: 10.1073/pnas.90.12.5414. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Dalgaard J. Z., Garrett R. A. Protein-coding introns from the 23S rRNA-encoding gene form stable circles in the hyperthermophilic archaeon Pyrobaculum organotrophum. Gene. 1992 Nov 2;121(1):103–110. doi: 10.1016/0378-1119(92)90167-n. [DOI] [PubMed] [Google Scholar]
  8. Deavin A., Mathias A. P., Rabin B. R. Mechanism of action of bovine pancreatic ribonuclease. Nature. 1966 Jul 16;211(5046):252–255. doi: 10.1038/211252a0. [DOI] [PubMed] [Google Scholar]
  9. Fitz-Gibbon S., Choi A. J., Miller J. H., Stetter K. O., Simon M. I., Swanson R., Kim U. J. A fosmid-based genomic map and identification of 474 genes of the hyperthermophilic archaeon Pyrobaculum aerophilum. Extremophiles. 1997 Feb;1(1):36–51. doi: 10.1007/s007920050013. [DOI] [PubMed] [Google Scholar]
  10. Gohda K., Oka K., Tomita K., Hakoshima T. Crystal structure of RNase T1 complexed with the product nucleotide 3'-GMP. Structural evidence for direct interaction of histidine 40 and glutamic acid 58 with the 2'-hydroxyl group of the ribose. J Biol Chem. 1994 Jul 1;269(26):17531–17536. doi: 10.2210/pdb1rls/pdb. [DOI] [PubMed] [Google Scholar]
  11. Heyduk T., Heyduk E., Severinov K., Tang H., Ebright R. H. Determinants of RNA polymerase alpha subunit for interaction with beta, beta', and sigma subunits: hydroxyl-radical protein footprinting. Proc Natl Acad Sci U S A. 1996 Sep 17;93(19):10162–10166. doi: 10.1073/pnas.93.19.10162. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Jensen T. H., Jensen A., Kjems J. Tools for the production and purification of full-length, N- or C-terminal 32P-labeled protein, applied to HIV-1 Gag and Rev. Gene. 1995 Sep 11;162(2):235–237. doi: 10.1016/0378-1119(95)00328-4. [DOI] [PubMed] [Google Scholar]
  13. Jensen T. H., Leffers H., Kjems J. Intermolecular binding sites of human immunodeficiency virus type 1 Rev protein determined by protein footprinting. J Biol Chem. 1995 Jun 9;270(23):13777–13784. doi: 10.1074/jbc.270.23.13777. [DOI] [PubMed] [Google Scholar]
  14. Kjems J., Garrett R. A. Novel splicing mechanism for the ribosomal RNA intron in the archaebacterium Desulfurococcus mobilis. Cell. 1988 Aug 26;54(5):693–703. doi: 10.1016/s0092-8674(88)80014-x. [DOI] [PubMed] [Google Scholar]
  15. Kjems J., Garrett R. A. Ribosomal RNA introns in archaea and evidence for RNA conformational changes associated with splicing. Proc Natl Acad Sci U S A. 1991 Jan 15;88(2):439–443. doi: 10.1073/pnas.88.2.439. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kjems J., Jensen J., Olesen T., Garrett R. A. Comparison of transfer RNA and ribosomal RNA intron splicing in the extreme thermophile and archaebacterium Desulfurococcus mobilis. Can J Microbiol. 1989 Jan;35(1):210–214. doi: 10.1139/m89-033. [DOI] [PubMed] [Google Scholar]
  17. Kjems J., Leffers H., Olesen T., Garrett R. A. A unique tRNA intron in the variable loop of the extreme thermophile Thermofilum pendens and its possible evolutionary implications. J Biol Chem. 1989 Oct 25;264(30):17834–17837. [PubMed] [Google Scholar]
  18. Kleman-Leyer K., Armbruster D. W., Daniels C. J. Properties of H. volcanii tRNA intron endonuclease reveal a relationship between the archaeal and eucaryal tRNA intron processing systems. Cell. 1997 Jun 13;89(6):839–847. doi: 10.1016/s0092-8674(00)80269-x. [DOI] [PubMed] [Google Scholar]
  19. Kurihara H., Nonaka T., Mitsui Y., Ohgi K., Irie M., Nakamura K. T. The crystal structure of ribonuclease Rh from Rhizopus niveus at 2.0 A resolution. J Mol Biol. 1996 Jan 19;255(2):310–320. doi: 10.1006/jmbi.1996.0025. [DOI] [PubMed] [Google Scholar]
  20. Lykke-Andersen J., Aagaard C., Semionenkov M., Garrett R. A. Archaeal introns: splicing, intercellular mobility and evolution. Trends Biochem Sci. 1997 Sep;22(9):326–331. doi: 10.1016/s0968-0004(97)01113-4. [DOI] [PubMed] [Google Scholar]
  21. Lykke-Andersen J., Garrett R. A., Kjems J. Protein footprinting approach to mapping DNA binding sites of two archaeal homing enzymes: evidence for a two-domain protein structure. Nucleic Acids Res. 1996 Oct 15;24(20):3982–3989. doi: 10.1093/nar/24.20.3982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Lykke-Andersen J., Garrett R. A. Structural characteristics of the stable RNA introns of archaeal hyperthermophiles and their splicing junctions. J Mol Biol. 1994 Nov 11;243(5):846–855. doi: 10.1006/jmbi.1994.1687. [DOI] [PubMed] [Google Scholar]
  23. Lykke-Andersen J., Thi-Ngoc H. P., Garrett R. A. DNA substrate specificity and cleavage kinetics of an archaeal homing-type endonuclease from Pyrobaculum organotrophum. Nucleic Acids Res. 1994 Nov 11;22(22):4583–4590. doi: 10.1093/nar/22.22.4583. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Michel F., Ferat J. L. Structure and activities of group II introns. Annu Rev Biochem. 1995;64:435–461. doi: 10.1146/annurev.bi.64.070195.002251. [DOI] [PubMed] [Google Scholar]
  25. Milligan J. F., Uhlenbeck O. C. Synthesis of small RNAs using T7 RNA polymerase. Methods Enzymol. 1989;180:51–62. doi: 10.1016/0076-6879(89)80091-6. [DOI] [PubMed] [Google Scholar]
  26. Oubridge C., Ito N., Evans P. R., Teo C. H., Nagai K. Crystal structure at 1.92 A resolution of the RNA-binding domain of the U1A spliceosomal protein complexed with an RNA hairpin. Nature. 1994 Dec 1;372(6505):432–438. doi: 10.1038/372432a0. [DOI] [PubMed] [Google Scholar]
  27. Peebles C. L., Gegenheimer P., Abelson J. Precise excision of intervening sequences from precursor tRNAs by a membrane-associated yeast endonuclease. Cell. 1983 Feb;32(2):525–536. doi: 10.1016/0092-8674(83)90472-5. [DOI] [PubMed] [Google Scholar]
  28. Phizicky E. M., Greer C. L. Pre-tRNA splicing: variation on a theme or exception to the rule? Trends Biochem Sci. 1993 Jan;18(1):31–34. doi: 10.1016/0968-0004(93)90085-2. [DOI] [PubMed] [Google Scholar]
  29. Rauhut R., Green P. R., Abelson J. Yeast tRNA-splicing endonuclease is a heterotrimeric enzyme. J Biol Chem. 1990 Oct 25;265(30):18180–18184. [PubMed] [Google Scholar]
  30. Scott W. G., Murray J. B., Arnold J. R., Stoddard B. L., Klug A. Capturing the structure of a catalytic RNA intermediate: the hammerhead ribozyme. Science. 1996 Dec 20;274(5295):2065–2069. doi: 10.1126/science.274.5295.2065. [DOI] [PubMed] [Google Scholar]
  31. Tange T. O., Jensen T. H., Kjems J. In vitro interaction between human immunodeficiency virus type 1 Rev protein and splicing factor ASF/SF2-associated protein, p32. J Biol Chem. 1996 Apr 26;271(17):10066–10072. doi: 10.1074/jbc.271.17.10066. [DOI] [PubMed] [Google Scholar]
  32. Thompson L. D., Daniels C. J. A tRNA(Trp) intron endonuclease from Halobacterium volcanii. Unique substrate recognition properties. J Biol Chem. 1988 Dec 5;263(34):17951–17959. [PubMed] [Google Scholar]
  33. Thompson L. D., Daniels C. J. Recognition of exon-intron boundaries by the Halobacterium volcanii tRNA intron endonuclease. J Biol Chem. 1990 Oct 25;265(30):18104–18111. [PubMed] [Google Scholar]
  34. Trotta C. R., Miao F., Arn E. A., Stevens S. W., Ho C. K., Rauhut R., Abelson J. N. The yeast tRNA splicing endonuclease: a tetrameric enzyme with two active site subunits homologous to the archaeal tRNA endonucleases. Cell. 1997 Jun 13;89(6):849–858. doi: 10.1016/s0092-8674(00)80270-6. [DOI] [PubMed] [Google Scholar]
  35. Weber U., Beier H., Gross H. J. Another heritage from the RNA world: self-excision of intron sequence from nuclear pre-tRNAs. Nucleic Acids Res. 1996 Jun 15;24(12):2212–2219. doi: 10.1093/nar/24.12.2212. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Westaway S. K., Belford H. G., Apostol B. L., Abelson J., Greer C. L. Novel activity of a yeast ligase deletion polypeptide. Evidence for GTP-dependent tRNA splicing. J Biol Chem. 1993 Feb 5;268(4):2435–2443. [PubMed] [Google Scholar]
  37. Wich G., Leinfelder W., Böck A. Genes for stable RNA in the extreme thermophile Thermoproteus tenax: introns and transcription signals. EMBO J. 1987 Feb;6(2):523–528. doi: 10.1002/j.1460-2075.1987.tb04784.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Wodak S. Y. The structure of cytidilyl(2',5')adenosine when bound to pancreatic ribonuclease S. J Mol Biol. 1977 Nov;116(4):855–875. doi: 10.1016/0022-2836(77)90275-3. [DOI] [PubMed] [Google Scholar]
  39. van Tol H., Gross H. J., Beier H. Non-enzymatic excision of pre-tRNA introns? EMBO J. 1989 Jan;8(1):293–300. doi: 10.1002/j.1460-2075.1989.tb03376.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES