Enzymatic Analysis of Reconstituted Archaeal Exosomes.
Archaeal exosome
Complex reconstitution
In vitro transcription
Protein complex
RNA degradation
RNA polyadenylation
RNA-binding protein
Sulfolobus
Journal
Methods in molecular biology (Clifton, N.J.)
ISSN: 1940-6029
Titre abrégé: Methods Mol Biol
Pays: United States
ID NLM: 9214969
Informations de publication
Date de publication:
2020
2020
Historique:
entrez:
27
11
2019
pubmed:
27
11
2019
medline:
15
12
2020
Statut:
ppublish
Résumé
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.
Identifiants
pubmed: 31768972
doi: 10.1007/978-1-4939-9822-7_4
doi:
Substances chimiques
Archaeal Proteins
0
RNA, Archaeal
0
RNA-Binding Proteins
0
RNA
63231-63-0
DNA Primase
EC 2.7.7.-
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
63-79Références
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