Purification of Eukaryotic Exoribonucleases Following Heterologous Expression in Bacteria and Analysis of Their Biochemical Properties by In Vitro Enzymatic Assays

  • Rafal TomeckiEmail author
  • Karolina Drazkowska
  • Antonina Krawczyk
  • Katarzyna Kowalska
  • Andrzej DziembowskiEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1259)


Exoribonucleases—among the other RNases—play a crucial role in the regulation of different aspects of RNA metabolism in the eukaryotic cell. To fully understand the exact mechanism of activity exhibited by such enzymes, it is crucial to determine their detailed biochemical properties, notably their substrate specificity and optimal conditions for enzymatic action. One of the most significant features of exoribonucleases is the direction of degradation of RNA substrates, which can proceed either from 5′-end to 3′-end or in the opposite way. Here, we present methods allowing the efficient production and purification of eukaryotic exoribonucleases, the preparation and labeling of various RNA substrates, and the biochemical characterization of exonucleolytic activity. We also explain how the exonucleolytic activity may be distinguished from that of endonucleases.

Key words

Exoribonuclease RNase RNA degradation RNA Oligonucleotide labeling Isotope Fluorescent dye Enzymatic activity Thin-layer chromatography Denaturing polyacrylamide gel 



This work was supported by the National Science Centre within the grant allocated to RT on the basis of the decision number DEC-2011/01/D/NZ1/03510 and through a grant awarded to RT by the National Centre for Research and Development within the LIDER program (LIDER/35/46/L-3/11/NCBR/2012). RT was the recipient of a scholarship for outstanding young scientists from the Polish Ministry of Science and Higher Education.


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Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Rafal Tomecki
    • 1
    • 2
    Email author
  • Karolina Drazkowska
    • 1
    • 2
  • Antonina Krawczyk
    • 2
    • 3
  • Katarzyna Kowalska
    • 1
    • 2
  • Andrzej Dziembowski
    • 1
    • 2
    Email author
  1. 1.Institute of Biochemistry and BiophysicsPolish Academy of SciencesWarsawPoland
  2. 2.Institute of Genetics and Biotechnology, Faculty of BiologyUniversity of WarsawWarsawPoland
  3. 3.Department of Molecular GeneticsGroningen Biomolecular Sciences and Biotechnology Institute, University of GroningenGroningenThe Netherlands

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