Assay of Bacillus subtilis Ribonuclease Activity In Vitro

Olivier Pellegrini; Laetitia Gilet; Aude Trinquier; Anastasia Tolcan; Delphine Allouche; Sylvain Durand; Frédérique Braun; Ciarán Condon

doi:10.1007/978-1-0716-0935-4_25

Assay of Bacillus subtilis Ribonuclease Activity In Vitro

Methods Mol Biol. 2021:2209:403-424. doi: 10.1007/978-1-0716-0935-4_25.

Authors

Olivier Pellegrini¹, Laetitia Gilet¹, Aude Trinquier¹, Anastasia Tolcan¹, Delphine Allouche¹, Sylvain Durand¹, Frédérique Braun¹, Ciarán Condon²

Affiliations

¹ Expression Génétique Microbienne, UMR8261, CNRS, Université de Paris, Institut de Biologie Physico-Chimique, Paris, France.
² Expression Génétique Microbienne, UMR8261, CNRS, Université de Paris, Institut de Biologie Physico-Chimique, Paris, France. condon@ibpc.fr.

PMID: 33201483
DOI: 10.1007/978-1-0716-0935-4_25

Abstract

Ribonucleases can cleave RNAs internally in endoribonucleolytic mode or remove one nucleotide at a time from either the 5' or 3' end through exoribonuclease action. To show direct implication of an RNase in a specific pathway of RNA maturation or decay requires the setting up of in vitro assays with purified enzymes and substrates. This chapter complements Chapter 24 on assays of ribonuclease action in vivo by providing detailed protocols for the assay of B. subtilis RNases with prepared substrates in vitro.

Keywords: In vitro transcription; Protein purification; RNA substrates; Radioactive labelling; Ribonuclease.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Bacillus subtilis / enzymology
Enzyme Assays / methods*
Gene Expression Regulation, Bacterial
Kinetics
RNA Probes / metabolism*
RNA, Bacterial / metabolism*
Ribonucleases / metabolism*

Substances

RNA Probes
RNA, Bacterial
Ribonucleases