Regulated transcriptional termination in bacteria was shown to control the expression of key metabolic and virulence related genes, as well as lysis/lysogeny decisions during phage infection. To uncover novel forms of regulated termination we developed a method for direct sequencing of RNA 3’ ends in bacteria. We demonstrate that our method can identify the vast majority of termination events in the cell in a genome wide manner and to the single nucleotide resolution. Using our approach we detected the majority of known riboswitches in B. subtilis in high sensitivity and specificity, and identified additional novel candidates regulating genes implicated in social behaviors such as swarming, antibiotic resistance and core metabolic processes. To characterize novel and known regulators, we devised an RNA-seq platform that allows unbiased, parallel in-vivo measurements of multiple riboswitch activities in different physiological conditions and enables high-throughput ligand screening. Our results provide the first experimental genome-wide method for analyzing termination events in bacteria and can be readily applied to discover novel regulators in any bacterial genome.
