In Vitro Study of the Major Bacillus subtilis Ribonucleases Y and J.

The metabolic instability of mRNA is fundamental to the adaptation of gene expression. In bacteria, mRNA decay follows first-order kinetics and is primarily controlled at the steps initiating degradation. In the model Gram-positive organism Bacillus subtilis, the major mRNA decay pathway initiates with an endonucleolytic cleavage by the membrane-associated RNase Y. High-throughput sequencing has identified a large number of potential mRNA substrates but our understanding of what parameters affect cleavage in vivo is still quite limited. In vitro reconstitution of the cleavage event is thus instrumental in defining the mechanistic details, substrate recognition, the role of auxiliary factors, and of membrane localization in cleavage. In this chapter, we describe not only the purification and assay of RNase Y but also RNase J1/J2 which shares a similar low-specificity endoribonucleolytic activity with RNase Y. We highlight potential problems in the set-up of these assays and include methods that allow purification of full-length RNase Y and its incorporation in multilamellar vesicles created from native B. subtilis lipids that might best mimic in vivo conditions.