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Enzymatic Analysis of Reconstituted Archaeal Exosomes

  • Elena Evguenieva-HackenbergEmail author
  • A. Susann Gauernack
  • Linlin Hou
  • Gabriele KlugEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2062)

Abstract

The archaeal exosome is a protein complex with phosphorolytic activity. It is built of a catalytically active hexameric ring containing the archaeal Rrp41 and Rrp42 proteins, and a heteromeric RNA-binding platform. The platform contains a heterotrimer of the archaeal Rrp4 and Csl4 proteins (which harbor S1 and KH or Zn-ribbon RNA binding domains), and comprises additional archaea-specific subunits. The latter are represented by the archaeal DnaG protein, which harbors a novel RNA-binding domain and tightly interacts with the majority of the exosome isoforms, and Nop5, known as a part of an rRNA methylating complex and found to associate with the archaeal exosome at late stationary phase. Although in the cell the archaeal exosome exists in different isoforms with heterotrimeric Rrp4-Csl4-caps, in vitro it is possible to reconstitute complexes with defined, homotrimeric caps and to study the impact of each RNA-binding subunit on exoribonucleolytic degradation and on polynucleotidylation of RNA. Here we describe procedures for reconstitution of isoforms of the Sulfolobus solfataricus exosome and for set-up of RNA degradation and polyadenylation assays.

Key words

Archaeal exosome In vitro transcription Protein complex Complex reconstitution RNA-binding protein RNA degradation RNA polyadenylation Sulfolobus 

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  1. 1.Institute for Microbiology and Molecular BiologyJustus-Liebig-University GiessenGiessenGermany

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