Investigation of sRNA-mRNA Interactions in Bacillus subtilis In Vivo.

In this chapter, we describe in vivo methods for the analysis of interactions between an sRNA and its target mRNA in B. subtilis. All these methods have been either established or significantly improved in our group and successfully employed to characterize a number of sRNA/target mRNA systems in Bacillus subtilis. Whereas in Chap. 8, we describe a combination of in vitro methods, e.g., EMSA and RNA secondary structure probing, we focus here on the investigation of RNA-RNA interactions in vivo using compatible plasmids or chromosomal insertions and deletions, the elucidation of the mechanisms of action of regulatory sRNAs employing transcriptional and translational reporter gene fusions, as well as the determination of expression profiles, half-lives of sRNA and mRNA, and their intracellular concentrations, and, finally, the investigation of RNA chaperones that promote the sRNA/mRNA interaction. For an in-depth analysis of sRNA-mRNA interactions in B. subtilis, a combination of in vivo and in vitro methods should be applied.