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Methods in molecular biology (Clifton, N.J.)
2020;2062 63-79.

Enzymatic Analysis of Reconstituted Archaeal Exosomes.

Elena Evguenieva-Hackenberg, A Susann Gauernack, Linlin Hou, Gabriele Klug
Author Information
  1. Elena Evguenieva-Hackenberg: Institute for Microbiology and Molecular Biology, Justus-Liebig-University Giessen, Giessen, Germany. Elena.Evguenieva-Hackenberg@mikro.bio.uni-giessen.de.
  2. A Susann Gauernack: Institute for Microbiology and Molecular Biology, Justus-Liebig-University Giessen, Giessen, Germany.
  3. Linlin Hou: Institute for Microbiology and Molecular Biology, Justus-Liebig-University Giessen, Giessen, Germany.
  4. Gabriele Klug: Institute for Microbiology and Molecular Biology, Justus-Liebig-University Giessen, Giessen, Germany. Gabriele.Klug@mikro.bio.uni-giessen.de.
PMID: 31768972 DOI: 10.1007/978-1-4939-9822-7_4

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.

Keywords

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

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MeSH Term

Archaeal Proteins
DNA Primase
Escherichia coli
Exosomes
Polyadenylation
RNA
RNA Stability
RNA, Archaeal
RNA-Binding Proteins
Sulfolobus solfataricus

Chemicals

Archaeal Proteins
RNA, Archaeal
RNA-Binding Proteins
RNA
DNA Primase
Journal Article
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Word Cloud

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