title: Purification of eukaryotic exoribonucleases following heterologous expression in bacteria and analysis of their biochemical properties by in vitro enzymatic assays.
abstract: Exoribonucleases-among the other RNases-play a crucial role in the regulation of different aspects of RNA metabolism in the eukaryotic cell. To fully understand the exact mechanism of activity exhibited by such enzymes, it is crucial to determine their detailed biochemical properties, notably their substrate specificity and optimal conditions for enzymatic action. One of the most significant features of exoribonucleases is the direction of degradation of RNA substrates, which can proceed either from 5'-end to 3'-end or in the opposite way. Here, we present methods allowing the efficient production and purification of eukaryotic exoribonucleases, the preparation and labeling of various RNA substrates, and the biochemical characterization of exonucleolytic activity. We also explain how the exonucleolytic activity may be distinguished from that of endonucleases.
authors.affiliation: Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawinskiego 5a, 02-106, Warsaw, Poland, rtom1916@gmail.com.
authors.forename: Rafal
authors.initials: R
authors.lastname: Tomecki
authors.forename: Karolina
authors.initials: K
authors.lastname: Drazkowska
authors.forename: Antonina
authors.initials: A
authors.lastname: Krawczyk
authors.forename: Katarzyna
authors.initials: K
authors.lastname: Kowalska
authors.forename: Andrzej
authors.initials: A
authors.lastname: Dziembowski
mesh_terms: D001419:Bacteria / Q000378:metabolism*; D002855:Chromatography, Thin Layer; D057075:Enzyme Assays / Q000379:methods*; D005095:Exoribonucleases / Q000302:isolation & purification* / Q000378:metabolism*; D012313:RNA
chemical_list: D012313:RNA; D005095:Exoribonucleases