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Archives of Microbiology

, 190:301 | Cite as

Two archaeal tRNase Z enzymes: similar but different

  • Bettina Späth
  • Sylvia Schubert
  • Annika Lieberoth
  • Florian Settele
  • Stefanie Schütz
  • Susan Fischer
  • Anita MarchfelderEmail author
Original Paper

Abstract

The endoribonuclease tRNase Z plays an essential role in tRNA metabolism by removal of the 3′ trailer element of precursor RNAs. To investigate tRNA processing in archaea, we identified and expressed the tRNase Z from Haloferax volcanii, a halophilic archaeon. The recombinant enzyme is a homodimer and efficiently processes precursor tRNAs. Although the protein is active in vivo at 2–4 M KCl, it is inhibited by high KCl concentrations in vitro, whereas 2–3 M (NH4)2SO4 do not inhibit tRNA processing. Analysis of the metal content of the metal depleted tRNase Z revealed that it still contains 0.4 Zn2+ ions per dimer. In addition tRNase Z requires Mn2+ ions for processing activity. We compared the halophilic tRNase Z to the homologous one from Pyrococcus furiosus, a thermophilic archaeon. Although both enzymes have 46% sequence similarity, they differ in their optimal reaction conditions. Both archaeal tRNase Z proteins process mitochondrial pre-tRNAs. Only the thermophilic tRNase Z shows in addition activity toward intron containing pre-tRNAs, 5′ extended precursors, the phosphodiester bis(p-nitrophenyl)phosphate (bpNPP) and the glyoxalase II substrate S-d-lactoylglutathion (SLG).

Keywords

Haloferax volcanii Pyrococcus furiosus tRNase Z tRNA Processing Metallo-β-lactamase 

Notes

Acknowledgments

We appreciate very much discussions about salt requirements of the halophilic enzyme with Prof. Dr. Richard Giége and his helpful advice in analysing processing with ammonium sulphate. We are grateful to Dr. Harald Huber and Prof. Dr. Karl. O. Stetter for the gift of P. furiosus cells. We like to thank Elli Bruckbauer and Ingrid Schleyer for expert technical assistance. Work presented here was funded by the VolkswagenStiftung.

Supplementary material

203_2008_368_MOESM1_ESM.tif (4.8 mb)
Supplementary Fig. 1 Alignment of tRNase Z protein sequences. tRNase Z protein sequences from Escherichia coli (Ecoz), Pyrococcus furiosus (Pfuz), Methanocaldococcus janaschii (Mjaz), Haloferax volcanii (Hvoz), and Arabidopsis thaliana (Athz1) were aligned to display conserved regions. Amino acids identical between at least three sequences are shadowed black, amino acids similar between at least three sequences are shadowed grey. Sequence similarity between PfuTrz and HvoTrz is 46%. (TIFF 4.76 MB)
203_2008_368_MOESM2_ESM.tif (1.7 mb)
Supplementary Fig. 2 Intron containing pre-tRNAs are substrates for the thermophilic tRNase Z enzyme. The intron containing archaeal pre-tRNAMet is processed by the thermophilic tRNase Z protein (lane P) yielding the tRNA with the intron (148 nucleotides) and the 3′ trailer (105 nucleotides), while the halophilic protein does not process this precursor (lane H). A DNA size marker is shown in lane m. Sizes are given in nucleotides at the left, precursor and products are shown schematically at the right. (TIFF 1.72 MB)
203_2008_368_MOESM3_ESM.doc (28 kb)
Supplementary Table 1 Observed rate constants for the archaeal tRNase Z enzymes. The rate constant kobs was determined for both tRNase Z enzymes HvoTrz and PfuTrz at an enzyme concentration of 3 and 5 pmol. (DOC 28.0 kb)

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Copyright information

© Springer-Verlag 2008

Authors and Affiliations

  • Bettina Späth
    • 5
  • Sylvia Schubert
    • 1
  • Annika Lieberoth
    • 2
  • Florian Settele
    • 3
  • Stefanie Schütz
    • 4
  • Susan Fischer
    • 5
  • Anita Marchfelder
    • 5
    Email author
  1. 1.Valckenburg SchuleUlmGermany
  2. 2.Zentrum für Molekulare Neurobiologie, Institut für Biosynthese neuraler StrukturenUniversität HamburgHamburgGermany
  3. 3.Department of Cell Cycle Control and Carcinogenesis, F045DKFZHeidelbergGermany
  4. 4.Department of General Zoology and EndocrinologyUniversität UlmUlmGermany
  5. 5.Biologie IIUniversität UlmUlmGermany

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