Purification of eukaryotic exoribonucleases following heterologous expression in bacteria and analysis of their biochemical properties by in vitro enzymatic assays.

Rafal Tomecki, Karolina Drazkowska, Antonina Krawczyk, Katarzyna Kowalska, Andrzej Dziembowski

Methods in molecular biology (Clifton, N.J.) 2015

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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.

Citation

Rafal Tomecki, Karolina Drazkowska, Antonina Krawczyk, Katarzyna Kowalska, Andrzej Dziembowski. Purification of eukaryotic exoribonucleases following heterologous expression in bacteria and analysis of their biochemical properties by in vitro enzymatic assays. Methods in molecular biology (Clifton, N.J.). 2015;1259:417-52