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Molecular Genetics and Genomics

, Volume 274, Issue 3, pp 235–247 | Cite as

Structural and functional analysis of 5S rRNA in Saccharomyces cerevisiae

  • Sergey Kiparisov
  • Alexey Petrov
  • Arturas Meskauskas
  • Petr V. Sergiev
  • Olga A. Dontsova
  • Jonathan D. Dinman
Original Paper

Abstract

5S rRNA extends from the central protuberance of the large ribosomal subunit, through the A-site finger, and down to the GTPase-associated center. Here, we present a structure-function analysis of seven 5S rRNA alleles which are sufficient for viability in the yeast Saccharomyces cerevisiae when expressed in the absence of wild-type 5S rRNAs, and extend this analysis using a large bank of mutant alleles that show semi-dominant phenotypes in the presence of wild-type 5S rRNA. This analysis supports the hypothesis that 5S rRNA serves to link together several different functional centers of the ribosome. Data are also presented which suggest that in eukaryotic genomes selection has favored the maintenance of multiple alleles of 5S rRNA, and that these may provide cells with a mechanism to post-transcriptionally regulate gene expression.

Keywords

5S rRNA Ribosome Virus Frameshifting Drugs 

Notes

Acknowledgements

We wish to thank the members of the Dinman and Dontsova laboratories, with special thanks to Sarah Fraser, Ewan Plant, Steve Hutcheson and Alexey Bogdanov for their advice and support. This work was supported by grants to JDD from the National Institutes of Health (GM62143), and to JDD and OAD from the Fogarty International Center (TW005787), and to OAD from HHMI 55000303 and RFBR.

References

  1. Altschul SF, Gish W, Miller E, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215:403–410CrossRefPubMedGoogle Scholar
  2. Ban N, Nissen P, Hansen J, Moore PB, Steitz TA (2000) The complete atomic structure of the large ribosomal subunit at 2.4Å resolution. Science 289:905–920CrossRefPubMedGoogle Scholar
  3. Betzel C, Lorenz S, Furste JP, Bald R, Zhang M, Schneider TR, Wilson KS, Erdmann VA (1994) Crystal structure of domain A of Thermus flavus 5S rRNA and the contribution of water molecules to its structure. FEBS Lett 351:159–164CrossRefPubMedGoogle Scholar
  4. Boeke JD, Xu H, Fink GR (1988) A general method for the chromosomal amplification of genes into yeast. Science 239:280–282PubMedCrossRefGoogle Scholar
  5. Bogdanov AA, Dontsova OA, Dokudovskaya SS, Lavrik IN (1995) Structure and function of 5S rRNA in the ribosome. Biochem Cell Biol 73:869–876PubMedCrossRefGoogle Scholar
  6. Brow DA, Geiduschek EP (1987) Modulation of yeast 5S rRNA synthesis in vitro by ribosomal protein YL3. J Biol Chem 262:13953–13958PubMedGoogle Scholar
  7. Carroll K, Wickner RB (1995) Translation and M1 dsRNA propagation: MAK18 = RPL41B and cycloheximide curing. J Bacteriol 177:2887–2891PubMedGoogle Scholar
  8. Christianson TW, Sikorski RS, Dante M, Shero JH, Hieter P (1992) Multifunctional yeast high-copy-number shuttle vectors. Yeast 110:119–122Google Scholar
  9. Deshmukh M, Tsay YF, Paulovich AG, Woolford JL, Jr (1993) Yeast ribosomal protein L1 is required for the stability of newly synthesized 5S rRNA and the assembly of 60S ribosomal subunits. Mol Cell Biol 13:2835–2845PubMedGoogle Scholar
  10. Deshmukh M, Stark J, Yeh LC, Lee JC, Woolford JL Jr (1995) Multiple regions of yeast ribosomal protein L1 are important for its interaction with 5S rRNA and assembly into ribosomes. J Biol Chem 270:30148–30156CrossRefPubMedGoogle Scholar
  11. Dinman JD (1995) Ribosomal frameshifting in yeast viruses. Yeast 11:1115–1127CrossRefPubMedGoogle Scholar
  12. Dinman JD, Wickner RB (1992) Ribosomal frameshifting efficiency and Gag/Gag-pol ratio are critical for yeast M1 double-stranded RNA virus propagation. J Virol 66:3669–3676PubMedGoogle Scholar
  13. Dinman JD, Wickner RB (1995) 5S rRNA is involved in fidelity of translational reading frame. Genetics 141:95–105PubMedGoogle Scholar
  14. Dinman JD, Icho T, Wickner RB (1991) A −1 ribosomal frameshift in a double-stranded RNA virus forms a Gag-pol fusion protein. Proc Natl Acad Sci USA 88:174–178PubMedCrossRefGoogle Scholar
  15. Dinman JD, Ruiz-Echevarria MJ, Czaplinski K, Peltz SW (1997) Peptidyl transferase inhibitors have antiviral properties by altering programmed −1 ribosomal frameshifting efficiencies: development of model systems. Proc Natl Acad Sci USA 94:6606–6611CrossRefPubMedGoogle Scholar
  16. Dokudovskaya S, Dontsova O, Shpanchenko O, Bogdanov A, Brimacombe R (1996) Loop IV of 5S ribosomal RNA has contacts both to domain II and to domain V of the 23S RNA. RNA 2:146–152PubMedGoogle Scholar
  17. Farabaugh PJ (1996) Programmed translational frameshifting. Microbiol Rev 60:103–134PubMedGoogle Scholar
  18. Ford PJ, Southern EM (1973) Different sequences for 5S RNA in kidney cells and ovaries of Xenopus laevis. Nat New Biol 241:7–12PubMedGoogle Scholar
  19. Frank J (2003) Electron microscopy of functional ribosome complexes. Biopolymers 68:223–233CrossRefPubMedGoogle Scholar
  20. Funari SS, Rapp G, Perbandt M, Dierks K, Vallazza M, Betzel C, Erdmann VA, Svergun DI (2000) Structure of free Thermus flavus 5S rRNA at 1.3 nm resolution from synchrotron X-ray solution scattering. J Biol Chem 275:31283–31288CrossRefPubMedGoogle Scholar
  21. Harger JW, Dinman JD (2003) An in vivo dual-luciferase assay system for studying translational recoding in the yeast Saccharomyces cerevisiae. RNA 9:1019–1024CrossRefPubMedGoogle Scholar
  22. Harger JW, Dinman JD (2004) Evidence against a direct role for the Upf proteins in frameshifting or nonsense codon readthrough. RNA 10:1721–1729CrossRefPubMedGoogle Scholar
  23. Harger JW, Meskauskas A, Dinman JD (2002) An ’integrated model’ of programmed ribosomal frameshifting and post-transcriptional surveillance. Trends Biochem Sci 27:448–454CrossRefPubMedGoogle Scholar
  24. Harms J, Schluenzen F, Zarivach R, Bashan A, Gat S, Agmon I, Bartels H, Franceschi F, Yonath A (2001) High resolution structure of the large ribosomal subunit from a mesophilic eubacterium. Cell 107:679–688CrossRefPubMedGoogle Scholar
  25. Huber PW, Rife JP, Moore PB (2001) The structure of helix III in Xenopus oocyte 5S rRNA: an RNA stem containing a two-nucleotide bulge. J Mol Biol 312:823–832CrossRefPubMedGoogle Scholar
  26. Ito H, Fukuda Y, Murata K, Kimura A (1983) Transformation of intact yeast cells treated with alkali cations. J Bacteriol 153:163–168PubMedGoogle Scholar
  27. Jacobs JL, Dinman JD (2004) Systematic analysis of bicistronic reporter assay data. Nucleic Acids Res 32:e160–e170CrossRefPubMedGoogle Scholar
  28. Kawakami K, Paned S, Faioa B, Moore DP, Boeke JD, Farabaugh PJ, Strathern JN, Nakamura Y, Garfinkel DJ (1993) A rare tRNA-Arg(CCU) that regulates Ty1 element ribosomal frameshifting is essential for Ty1 retrotransposition in Saccharomyces cerevisiae. Genetics 135:309–320PubMedGoogle Scholar
  29. Kinzy TG, Harger JW, Carr-Schmid A, Kwon J, Shastry M, Justice MC, Dinman JD (2002) New targets for antivirals: the ribosomal A-site and the factors that interact with it. Virology 300:60–70CrossRefPubMedGoogle Scholar
  30. Lorenz S, Perbandt M, Lippmann C, Moore K, DeLucas LJ, Betzel C, Erdmann VA (2000) Crystallization of engineered Thermus flavus 5S rRNA under earth and microgravity conditions. Acta Crystallogr D Biol Crystallogr 56:498–500CrossRefPubMedGoogle Scholar
  31. Meskauskas A, Dinman JD (2001) Ribosomal protein L5 helps anchor peptidyl-tRNA to the P-site in Saccharomyces cerevisiae. RNA 7:1084–1096CrossRefPubMedGoogle Scholar
  32. Meskauskas A, Baxter JL, Carr EA, Yasenchak J, Gallagher JEG, Baserga SJ, Dinman JD (2003a) Delayed rRNA processing results in significant ribosome biogenesis and functional defects. Mol Cell Biol 23:1602–1613CrossRefPubMedGoogle Scholar
  33. Meskauskas A, Harger JW, Jacobs KLM, Dinman JD (2003b) Decreased peptidyltransferase activity correlates with increased programmed −1 ribosomal frameshifting and viral maintenance defects in the yeast Saccharomyces cerevisiae. RNA 9:982–992CrossRefPubMedGoogle Scholar
  34. Noller HF, Yusupov MM, Yusupova GZ, Baucom A, Lieberman K, Lancaster L, Dallas A, Fredrick K, Earnest TN, Cate JH (2001) Structure of the ribosome at 5.5Å resolution and its interactions with functional ligands. Cold Spring Harb Symp Quant Biol 66:57–66CrossRefPubMedGoogle Scholar
  35. Oakes M, Aris JP, Brockenbrough JS, Wai H, Vu L, Nomura M (1998) Mutational analysis of the structure and localization of the nucleolus in the yeast Saccharomyces cerevisiae. J Cell Biol 143:23–34CrossRefPubMedGoogle Scholar
  36. Ogle JM, Ramakrishnan V (2005) Structural insights into translational fidelity. Annu Rev Biochem 74:129–177CrossRefPubMedGoogle Scholar
  37. Ohtake Y, Wickner RB (1995a) KRB1, a suppressor of mak7-1 (a mutant RPL4A), is RPL4B, a second ribosomal protein L4 gene, on a fragment of Saccharomyces chromosome XII. Genetics 140:129–137PubMedGoogle Scholar
  38. Ohtake Y, Wickner RB (1995b) Yeast virus propagation depends critically on free 60S ribosomal subunit concentration. Mol Cell Biol 15:2772–2781PubMedGoogle Scholar
  39. Pestka S (1977) Inhibitors of protein synthesis. In: Weissbach H, Pestka S (eds) Molecular mechanisms of protein biosynthesis. Academic, New York, pp 467–553Google Scholar
  40. Petes TD (1979a) Meiotic mapping of yeast ribosomal deoxyribonucleic acid on chromosome XII. J Bacteriol 138:185–192PubMedGoogle Scholar
  41. Petes TD (1979b) Yeast ribosomal DNA genes are located on chromosome XII. Proc Natl Acad Sci USA 76:410–414PubMedCrossRefGoogle Scholar
  42. Petrov A, Meskauskas A, Dinman JD (2004) Ribosomal protein L3: influence on ribosome structure and function. RNA Biol 1:59–65PubMedGoogle Scholar
  43. Rose MD, Winston F, Hieter P (1990) Methods in yeast genetics. Cold Spring Harbor Laboratory Press, Cold Spring HarborGoogle Scholar
  44. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning, a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring HarborGoogle Scholar
  45. Sigmund CD, Ettayebi M, Borden A, Morgan EA (1988) Antibiotic resistance mutations in ribosomal RNA genes of Escherichia coli. Methods Enzymol 164:673–690PubMedCrossRefGoogle Scholar
  46. Smith MW, Meskauskas A, Wang P, Sergiev PV, Dinman JD (2001) Saturation mutagenesis of 5S rRNA in Saccharomyces cerevisiae. Mol Cell Biol 21:8264–8275CrossRefPubMedGoogle Scholar
  47. Sommer SS, Wickner RB (1982) Co-curing of plasmids affecting killer double-stranded RNAs of Saccharomyces cerevisiae: [HOK], [NEX], and the abundance of L are related and further evidence that M1 requires L. J Bacteriol 150:545–551PubMedGoogle Scholar
  48. Spahn CM, Beckmann R, Eswar N, Penczek PA, Sali A, Blobel G, Frank J (2001) Structure of the 80S ribosome from Saccharomyces cerevisiae—tRNA-ribosome and subunit-subunit interactions. Cell 107:373–386CrossRefPubMedGoogle Scholar
  49. Spahn CM, Gomez-Lorenzo MG, Grassucci RA, Jorgensen R, Andersen GR, Beckmann R, Penczek PA, Ballesta JP, Frank J (2004) Domain movements of elongation factor eEF2 and the eukaryotic 80S ribosome facilitate tRNA translocation. EMBO J 23:1008–1019CrossRefPubMedGoogle Scholar
  50. Steitz TA, Moore PB (2003) RNA, the first macromolecular catalyst: the ribosome is a ribozyme. Trends Biochem Sci 28:411–418CrossRefPubMedGoogle Scholar
  51. Stern S, Moazed D, Noller HF (1988) Structural analysis of RNA using chemical and enzymatic probing monitored by primer extension. Methods Enzymol 164:481–489PubMedGoogle Scholar
  52. Szymanski M, Barciszewska MZ, Erdmann VA, Barciszewski J (2002) 5S Ribosomal RNA Database. Nucleic Acids Res 30:176–178CrossRefPubMedGoogle Scholar
  53. Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680PubMedCrossRefGoogle Scholar
  54. Velichutina IV, Hong JY, Mesecar AD, Chernoff YO, Liebman SW (2001) Genetic interaction between yeast Saccharomyces cerevisiae release factors and the decoding region of 18S rRNA. J Mol Biol 305:715–727CrossRefPubMedGoogle Scholar
  55. Wai HH, Vu L, Oakes M, Nomura M (2000) Complete deletion of yeast chromosomal rDNA repeats and integration of a new rDNA repeat: use of rDNA deletion strains for functional analysis of rDNA promoter elements in vivo. Nucleic Acids Res 28:3524–3534CrossRefPubMedGoogle Scholar
  56. Wickner RB (1986) Double-stranded RNA replication in the yeast: the killer system. Annu Rev Biochem 55:373–395CrossRefPubMedGoogle Scholar
  57. Wickner RB (1996) Double-stranded RNA viruses of Saccharomyces cerevisiae. Microbiol Rev 60:250–265PubMedGoogle Scholar
  58. Wickner RB, Leibowitz MJ (1976) Two chromosomal genes required for killing expression in killer strains of Saccharomyces cerevisiae. Genetics 82:429–442PubMedGoogle Scholar
  59. Wickner RB, Porter-Ridley S, Fried HM, Ball SG (1982) Ribosomal protein L3 is involved in replication or maintenance of the killer double-stranded RNA genome of Saccharomyces cerevisiae. Proc Natl Acad Sci USA 79:4706–4708PubMedCrossRefGoogle Scholar
  60. Wilson DN, Nierhaus KH (2003) The ribosome through the looking Glass. Angew Chem Int Ed Engl 42:3464–3486CrossRefPubMedGoogle Scholar
  61. Xiong Y, Sundaralingam M (2000) Two crystal forms of helix II of Xenopus laevis 5S rRNA with a cytosine bulge. RNA 6:1316–1324CrossRefPubMedGoogle Scholar
  62. Yeh LC, Lee JC (1995) An in vitro system for studying RNA-protein interaction: application to a study of yeast ribosomal protein L1 binding to 5S rRNA. Biochimie 77:167–173CrossRefPubMedGoogle Scholar
  63. Yonath A et al (1998) Crystallographic studies on the ribosome, a large macromolecular assembly exhibiting severe nonisomorphism, extreme beam sensitivity and no internal symmetry. Acta Crystallogr A 54:945–955CrossRefPubMedGoogle Scholar
  64. Yusupov MM, Yusupova GZ, Baucom A, Lieberman K, Earnest TN, Cate JH, Noller HF (2001) Crystal structure of the ribosome at 5.5Å resolution. Science 292:883–896CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag 2005

Authors and Affiliations

  • Sergey Kiparisov
    • 1
  • Alexey Petrov
    • 2
  • Arturas Meskauskas
    • 2
  • Petr V. Sergiev
    • 1
  • Olga A. Dontsova
    • 1
  • Jonathan D. Dinman
    • 2
  1. 1.Department of ChemistryMoscow State UniversityMoscowRussia
  2. 2.Department of Cell Biology and Molecular GeneticsUniversity of MarylandCollege ParkUSA

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