In Vitro Characterization of the Prokaryotic Counterparts of the Exosome Complex
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.
Key wordsExoribonuclease PNPase RNase R RNase II EMSA Electrophoresis
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).