Ribosomal RNA N-glycosylase Activity Assay of Ribosome-inactivating Proteins

Rosario Iglesias; Lucía Citores; José M Ferreras

doi:10.21769/BioProtoc.2180

Ribosomal RNA N-glycosylase Activity Assay of Ribosome-inactivating Proteins

Bio Protoc. 2017 Mar 20;7(6):e2180. doi: 10.21769/BioProtoc.2180.

Authors

Rosario Iglesias¹, Lucía Citores¹, José M Ferreras¹

Affiliation

¹ Department of Biochemistry and Molecular Biology and Physiology, Faculty of Sciences, University of Valladolid, Valladolid, Spain.

Abstract

Ribosome-inactivating proteins (RIPs) are enzymes that irreversibly inactivate ribosomes as a consequence of their N-glycosylase (EC 3.2.2.22) activity. The enzyme cleaves the N-glycosidic bond between the adenine No. 4324 from the 28S rRNA and its ribose in rat ribosomes (or the equivalent adenine in sensitive ribosomes from other organisms). This adenine is located in the α-sarcin-ricin loop (SRL) that is crucial for anchoring the elongation factor (EF) G and EF2 on the ribosome during mRNA-tRNA translocation in prokaryotes and eukaryotes, respectively. RIPs have been isolated mainly from plants and examples of these proteins are ricin or Pokeweed Antiviral Protein (PAP). These proteins, either alone or as a part of immunotoxins, are useful tools for cancer therapy. The following protocol describes a method to detect the RNA fragment released when the RIP-treated apurinic RNA from rabbit reticulocyte lysate is incubated in the presence of acid aniline by electrophoresis on polyacrylamide gels. The fragment released (Endo's fragment) is diagnostic of the action of RIPs.

Keywords: Beetin 27; Pokeweed Antiviral Protein (PAP); Polynucleotide:adenosine glycosylase; Protein synthesis (inhibition); Ribosome-inactivating protein (RIP); Ricin; Sarcin-ricin loop; rRNA N-glycosylase.