Molecular Genetics and Genomics

, Volume 280, Issue 4, pp 337–350

Functional features of the C-terminal region of yeast ribosomal protein L5

  • Hossein Moradi
  • Ivailo Simoff
  • Galyna Bartish
  • Odd Nygård
Original Paper

Abstract

The aim of this study was to analyze the functional importance of the C-terminus of the essential yeast ribosomal protein L5 (YrpL5). Previous studies have indicated that the C-terminal region of YrpL5 forms an α-helix with a positively charged surface that is involved in protein–5S rRNA interaction. Formation of an YrpL5·5S rRNA complex is a prerequisite for nuclear import of YrpL5. Here we have tested the importance of the α-helix and the positively charged surface for YrpL5 function in Saccharomyces cerevisiae using site directed mutagenesis in combination with functional complementation. Alterations in the sequence forming the putative α-helix affected the functional capacity of YrpL5. However, the effect did not correlate with a decreased ability of the protein to bind to 5S rRNA as all rpL5 mutants tested were imported to the nucleus whether or not the α-helix or the positively charged surface were intact. The alterations introduced in the C-terminal sequence affected the growth rate of cells expressing mutant but functional forms of YrpL5. The reduced growth rate was correlated with a reduced ribosomal content per cell indicating that the alterations introduced in the C-terminus interfered with ribosome assembly.

Keywords

Functional complementation Mutation analysis Ribosomal protein L5 S. cerevisiae 

Abbreviations

YrpL5

Yeast ribosomal protein L5

Gal

Galactose

Glu

Glucose

SC

Synthetic complete

5FOA

5-Fluorortic acid

RNP

Ribonucleoprotein

SDS

Sodium dodecyl sulphate

Supplementary material

438_2008_369_MOESM1_ESM.pdf (152 kb)
MOESM1 [INSERT CAPTION HERE] (PDF 151 kb)

References

  1. Bartish G, Moradi H, Nygård O (2007) Amino acids Thr56 and Thr58 are not essential for elongation factor 2 function in yeast. FEBS J 274:5285–5297PubMedCrossRefGoogle Scholar
  2. Bellemare G, Vigne R, Jordan BR (1973) Interaction between Escherichia coli ribosomal proteins and 5S RNA molecules: recognition of prokaryotic 5S RNAs and rejection of eukaryotic 5S RNAs. Biochimie 55:29–35PubMedCrossRefGoogle Scholar
  3. 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
  4. Claussen M, Rudt F, Pieler T (1999) Functional modules in ribosomal protein L5 for ribonucleoprotein complex formation and nucleocytoplasmic transport. J Biol Chem 274:33951–33958PubMedCrossRefGoogle Scholar
  5. Combet C, Blanchet C, Geourjon C, Deleage G (2000) NPS@: network protein sequence analysis. Trends Biochem Sci 25:147–150PubMedCrossRefGoogle Scholar
  6. Deshmukh M, Tsay YF, Paulovich AG, Woolford JLJ (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
  7. Deshmukh M, Stark J, Yeh LC, Lee JC, Woolford JLJ (1995) Multiple regions of yeast ribosomal protein L1 are important for its interaction with 5 S rRNA and assembly into ribosomes. J Biol Chem 270:30148–30156PubMedCrossRefGoogle Scholar
  8. DiNitto JP, Huber PW (2003) Mutual induced fit binding of Xenopus ribosomal protein L5 to 5S rRNA. J Mol Biol 330:979–992PubMedCrossRefGoogle Scholar
  9. Draper DE, Reynaldo LP (1999) RNA binding strategies of ribosomal proteins. Nucleic Acids Res 27:381–388PubMedCrossRefGoogle Scholar
  10. Fox GW, Woese CR (1975) 5S RNA secondary structure. Nature 256:505–507PubMedCrossRefGoogle Scholar
  11. Iwasaki K, Kikukawa S, Kawamura S, Kouzuma Y, Tanaka I, Kimura M (2002) On the interaction of ribosomal protein L5 with 5S rRNA. Biosci Biotechnol Biochem 66:103–109PubMedCrossRefGoogle Scholar
  12. Kimata Y, Kohno K (1994) Elongation factor 2 mutants deficient in diphthamide formation show temperature-sensitive cell growth. J Biol Chem 269:13497–13501PubMedGoogle Scholar
  13. Koch AL, Deppe CS (1971) In vivo assay of protein synthesizing capacity of Escherichia coli from slowly growing chemostat cultures. J Mol Biol 55:549–562PubMedCrossRefGoogle Scholar
  14. Koch AL, Koch AL, Deppe CS (1971) The adaptive responses of Escherichia coli to a feast and famine existence. Adv Microb Physiol 6:147–217PubMedCrossRefGoogle Scholar
  15. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685PubMedCrossRefGoogle Scholar
  16. Lam YW, Lamond AI, Mann M, Andersen JS (2007) Analysis of nucleolar protein dynamics reveals the nuclear degradation of ribosomal proteins. Curr Biol 17:749–760PubMedCrossRefGoogle Scholar
  17. Lin E, Lin SW, Lin A (2001) The participation of 5S rRNA in the co-translational formation of a eukaryotic 5S ribonucleoprotein complex. Nucleic Acids Res 29:2510–2516PubMedCrossRefGoogle Scholar
  18. Luehrsen KR, Fox GE (1981) Secondary structure of eukaryotic cytoplasmic 5S ribosomal RNA. Proc Natl Acad Sci USA 78:2150–2154PubMedCrossRefGoogle Scholar
  19. Luoma GA, Marshall AG (1978) Lasar Raman evidence for a new cloverleaf secondary structure for eucaryotic 5S RNA. J Mol Biol 125:95–105PubMedCrossRefGoogle Scholar
  20. Meskauskas A, Dinman JD (2001) Ribosomal protein L5 helps anchor peptidyl-tRNA to the P-site in Saccharomyces cerevisiae. RNA 7:1084–1096PubMedCrossRefGoogle Scholar
  21. Michael WM, Dreyfuss G (1996) Distinct domains in ribosomal protein L5 mediate 5 S rRNA binding and nucleolar localization. J Biol Chem 271:11571–11574PubMedCrossRefGoogle Scholar
  22. Nazar RN, Wildeman AG (1983) Three helical domains form a protein binding site in the 5S RNA–protein complex from eukaryotic ribosomes. Nucleic Acids Res 11:3155–3168PubMedCrossRefGoogle Scholar
  23. Nazar RN, Yaguchi M, Willick GE, Rollin CF, Roy C (1979) The 5-S RNA binding protein from yeast (Saccharomyces cerevisiae) ribosomes. Evolution of the eukaryotic 5-S RNA binding protein. Eur J Biochem 102:573–582PubMedCrossRefGoogle Scholar
  24. Nazar RN, Yaguchi M, Willick GE (1982) The 5S RNA–protein complex from yeast: a model for the evolution and structure of the eukaryotic ribosome. Can J Biochem 60:490–496PubMedCrossRefGoogle Scholar
  25. Neidhardt FC, Magasanik B (1960) Studies on the role of ribonucleic acid in the growth of bacteria. Biochim Biophys Acta 42:99–116PubMedCrossRefGoogle Scholar
  26. Nygard O, Nilsson L (1984) Quantification of the different ribosomal phases during the translational elongation cycle in rabbit reticulocyte lysates. Eur J Biochem 145:345–350PubMedCrossRefGoogle Scholar
  27. Ortiz PA, Kinzy TG (2005) Dominant-negative mutant phenotypes and the regulation of translation elongation factor 2 levels in yeast. Nucleic Acids Res 33:5740–5748PubMedCrossRefGoogle Scholar
  28. Perederina A, Nevskaya N, Nikonov O, Nikulin A, Dumas P, Yao M, Tanaka I, Garber M, Gongadze G, Nikonov S (2002) Detailed analysis of RNA–protein interactions within the bacterial ribosomal protein L5/5S rRNA complex. RNA 8:1548–1557PubMedGoogle Scholar
  29. Rosorius D, Fries B, Stauber RH, Hirschmann N, Bevec D, Hauber J (2000) Human ribosomal protein L5 contains defined nuclear localization and export signals. J Biol Chem 275:12061–12068PubMedCrossRefGoogle Scholar
  30. Ross JF, Orlowski M (1982) Growth-rate-dependent adjustment of ribosome function in chemostat-grown cells of the fungus Mucor racemosus. J Bacteriol 149:650–653PubMedGoogle Scholar
  31. Saccharomyces Genome Deletion Project. http://www-sequence.stanford.edu/group/yeast_deletion_project/spo.html. Cited 21 Aug 2008
  32. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, New YorkGoogle Scholar
  33. Schaechter M, Maaloe O, Kjeldgaard NO (1958) Dependency on medium and temperature of cell size and chemical composition during balanced grown of Salmonella typhimurium. J Gen Microbiol 19:592–606PubMedGoogle Scholar
  34. Schimmang T, Tollervey D, Kern H, Frank R, Hurt EC (1989) A yeast nucleolar protein related to mammalian fibrillarin is associated with small nucleolar RNA and is essential for viability. EMBO J 8:4015–4024PubMedGoogle Scholar
  35. Soni R, Carmichael JP, Murray JA (1993) Parameters affecting lithium acetate-mediated transformation of Saccharomyces cerevisiae and development of a rapid and simplified procedure. Curr Genet 24:455–459PubMedCrossRefGoogle Scholar
  36. 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 107:373–386PubMedCrossRefGoogle Scholar
  37. 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 23:1008–1019PubMedCrossRefGoogle Scholar
  38. Steitz JA, Berg C, Hendrick JP, La Branche-Chabot H, Metspalu A, Rinke J, Yario T (1988) A 5S rRNA/L5 complex is a precursor to ribosome assembly in mammalian cells. J Cell Biol 106:545–556PubMedCrossRefGoogle Scholar
  39. Szymanski M, Barciszewska MZ, Erdmann VA, Barciszewska J (2003) 5S rRNA: structure and interactions. Biochem J 371:641–651PubMedCrossRefGoogle Scholar
  40. Tang BZ, Nazar RN (1991) Structure of the yeast ribosomal 5 S RNA-binding protein YL3. J Biol Chem 266:6120–6123PubMedGoogle Scholar
  41. Timmers AC, Stuger R, Schaap PJ, van ‘t Riet J, Raue HA (1999) Nuclear and nucleolar localization of Saccharomyces cerevisiae ribosomal proteins S22 and S25. FEBS Lett 452:335–340Google Scholar
  42. Wagner M, Price G, Rothstein R (2006) The absence of Top3 reveals an interaction between the Sgs1 and Pif1 DNA helicases in Saccharomyces cerevisiae. Genetics 174:555–573PubMedCrossRefGoogle Scholar
  43. Wrede P, Erdmann VA (1973) Activities of B. stearothermophilus 50 S ribosomes reconstituted with prokaryotic and eukaryotic 5 S RNA. FEBS Lett 33:315–319PubMedCrossRefGoogle Scholar
  44. Yaguchi M, Rollin CF, Roy C, Nazar RN (1984) The 5S RNA binding protein from yeast (Saccharomyces cerevisiae) ribosomes. An RNA binding sequence in the carboxyl-terminal region. Eur J Biochem 139:451–457PubMedCrossRefGoogle Scholar
  45. Yeh LC, Lee JC (1990) Probing the yeast 5 S RNA–protein complex by fluorescence and controlled proteolytic digestion. Arch Biochem Biophys 276:481–485PubMedCrossRefGoogle Scholar
  46. Yeh LC, Lee JC (1995a) Contributions of multiple basic amino acids in the C-terminal region of yeast ribosomal protein L1 to 5 S rRNA binding and 60 S ribosome stability. J Mol Biol 246:295–307PubMedCrossRefGoogle Scholar
  47. Yeh LC, Lee JC (1995b) An in vitro system for studying RNA–protein interaction: application to a study of yeast ribosomal protein L1 binding to 5S rRNA. Biochimie 77:167–173PubMedCrossRefGoogle Scholar
  48. Yeh LC, Lee JC (1995c) Involvement of multiple basic amino acids in yeast ribosomal protein L1 in 5S rRNA recognition. Nucleic Acids Symp Ser 33:63–65Google Scholar
  49. Yeh LC, Horowitz PM, Lee JC (1988) Studies of RNA–protein interactions in the yeast 5 S ribonucleoprotein particles by fluorescence and tritium exchange. Implications for ribosomal assembly. J Biol Chem 263:17412–17417PubMedGoogle Scholar
  50. Yeh LC, Deshmukh M, Woolford JL, Lee JC (1996) Involvement of lysine 270 and lysine 271 of yeast 5S rRNA binding protein in RNA binding and ribosome assembly. Biochim Biophys Acta 1308:133–141PubMedGoogle Scholar
  51. Yu RS, Wittmann HG (1973) The sequence of steps in the attachment of 5-S RNA to cores of Escherichia coli ribosomes. Biochim Biophys Acta 324:375–385PubMedGoogle Scholar
  52. Zhang J, Harnpicharnchai P, Jakovljevic J, Tang L, Guo Y, Oeffinger M, Rout MP, Hiley SL, Hughes T, Woolford JL Jr (2007) Assembly factors Rpf2 and Rrs1 recruit 5S rRNA and ribosomal proteins rpL5 and rpL11 into nascent ribosomes. Genes Dev 21:2580–2592PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2008

Authors and Affiliations

  • Hossein Moradi
    • 1
    • 2
  • Ivailo Simoff
    • 1
    • 2
  • Galyna Bartish
    • 1
    • 2
  • Odd Nygård
    • 2
  1. 1.Department of Cell Biology, Arrhenius Laboratories E5Stockholm UniversityStockholmSweden
  2. 2.Cell Biology Unit, Department of Life SciencesSödertörns högskolaHuddingeSweden

Personalised recommendations