Abstract
The same basic set of enzymatic activities exhibited by the eukaryotic RNA exosome are also found in prokaryotes. Bacteria have two predominant and distinct 3′→5′ exoribonuclease activities: one is characterized by processive hydrolysis, derived from RNase II and RNase R, and the other by processive phosphorolysis, derived from PNPase. In this chapter we describe methods for (1) the overexpression and purification of these three proteins; and (2) their in vitro biochemical and enzymatic characterization—including RNA binding. The labeling and preparation of a set of specific RNA substrates is also described.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Arraiano CM et al (2010) The critical role of RNA processing and degradation in the control of gene expression. FEMS Microbiol Rev 34:883–923
Cairrao F, Chora A, Zilhao R, Carpousis AJ, Arraiano CM (2001) RNase II levels change according to the growth conditions: characterization of gmr, a new Escherichia coli gene involved in the modulation of RNase II. Mol Microbiol 39:1550–1561
Amblar M, Barbas A, Gomez-Puertas P, Arraiano CM (2007) The role of the S1 domain in exoribonucleolytic activity: substrate specificity and multimerization. RNA 13:317–327
Matos RG, Barbas A, Arraiano CM (2009) RNase R mutants elucidate the catalysis of structured RNA: RNA-binding domains select the RNAs targeted for degradation. Biochem J 423:291–301
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254
Casinhas J, Matos RG, Haddad N, Arraiano CM (2018) Biochemical characterization of Campylobacter jejuni PNPase, an exoribonuclease important for bacterial pathogenicity. Biochimie 147:70–79
Matos RG, Barbas A, Arraiano CM (2010) Comparison of EMSA and SPR for the characterization of RNA-RNase II complexes. Protein J 29:394–397
Barbas A, Matos RG, Amblar M, Lopez-Vinas E, Gomez-Puertas P, Arraiano CM (2008) New insights into the mechanism of RNA degradation by ribonuclease II: identification of the residue responsible for setting the RNase II end product. J Biol Chem 283:13070–13076
Barbas A, Matos RG, Amblar M, Lopez-Vinas E, Gomez-Puertas P, Arraiano CM (2009) Determination of key residues for catalysis and RNA cleavage specificity: one mutation turns RNase II into a “SUPER-ENZYME”. J Biol Chem 284:20486–20498
Lopez PJ, Marchand I, Joyce SA, Dreyfus M (1999) The C-terminal half of RNase E, which organizes the Escherichia coli degradosome, participates in mRNA degradation but not rRNA processing in vivo. Mol Microbiol 33:188–199
Arraiano CM, Barbas A, Amblar M (2008) Characterizing ribonucleases in vitro examples of synergies between biochemical and structural analysis. Methods Enzymol 447:131–160
Schaeffer D et al (2009) The exosome contains domains with specific endoribonuclease, exoribonuclease and cytoplasmic mRNA decay activities. Nat Struct Mol Biol 16:56–62
Grunberg-Manago M (1963) Polynucleotide phosphorylase. Prog Nucleic Acid Res Mol Biol 1:93–133
Littauer UZ, Soreq H (1982) Polynucleotide phosphorylase. Enzymes 15:517–553
Acknowledgments
This work was supported by project Lisboa-01-0145-FEDER-007660 (Microbiologia Molecular, Estrutural e Celular) funded by FEDER through COMPETE 2020—Programa Operacional Competitividade e Internacionalização (POCI) and by project PTDC/BIA-MIC/1399/2014 to CMA, project PTDC/BIM-MEC/3749/2014 to SCV and project PTDC/BIA-BQM/28479/2017 to RGM funded by Fundação para a Ciência e Tecnologia, Portugal (FCT). RGM was financed by an FCT contract (ref. CEECIND/02065/2017); SCV was financed by program FCT program IF (ref. IF/00217/2015).
Author information
Authors and Affiliations
Corresponding authors
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2020 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Matos, R.G., Viegas, S.C., Arraiano, C.M. (2020). In Vitro Characterization of the Prokaryotic Counterparts of the Exosome Complex. In: LaCava, J., Vaňáčová, Š. (eds) The Eukaryotic RNA Exosome. Methods in Molecular Biology, vol 2062. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9822-7_3
Download citation
DOI: https://doi.org/10.1007/978-1-4939-9822-7_3
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-4939-9821-0
Online ISBN: 978-1-4939-9822-7
eBook Packages: Springer Protocols