High-resolution RNA 3’-ends mapping of bacterial rho-dependent transcripts

Transcription termination in bacteria can occur either via rho-dependent or independent mechanisms. Rho-independent terminators are composed of a stem-loop RNA structure followed by a uridine stretch and are known to terminate in a precise manner. In contrast, rho-dependent terminators have loosely defined characteristics and are thought to terminate in a fuzzy manner. While transcripts ending in a rho-independent terminator are protected from 3’-5’ exonuclease digestion due to the stem-loop structure of the terminator, it remains unclear what protects rho-dependent genes from being degraded. In this study, we mapped the exact steady-state RNA 3’ ends of hundreds of E. coli genes terminated either by rho-dependent or independent mechanisms. We found that transcripts generated from rho-dependent termination have precise 3’-ends at steady state. These termini were localized immediately downstream of energetically stable stem-loop structures, which were not followed by uridine rich sequences. We provide evidence that these structures protect rho-dependent genes from 3’-5’ exonucleases such as PNPase and RNase II, and present data localizing the Rho-utilization (rut) sites immediately downstream of these protective structures. This study represents the first extensive in-vivo map of exact RNA 3’-ends of rho-dependent transcripts in E. coli.