Engineering Transcriptional Interference through RNA Polymerase Processivity Control.

Antisense transcription is widespread in all kingdoms of life and has been shown to influence gene expression through transcriptional interference (TI), a phenomenon in which one transcriptional process negatively influences another in cis. The processivity, or uninterrupted transcription, of an RNA polymerase (RNAP) is closely tied to levels of antisense transcription in bacterial genomes, but its influence on TI, while likely important, is not well-characterized. Here, we show that TI can be tuned through processivity control via three distinct antitermination strategies: the antibiotic bicyclomycin, phage protein Psu, and ribosome-RNAP coupling. We apply these methods toward TI and tune ribosome-RNAP coupling to produce 38-fold transcription-level gene repression due to both RNAP collisions and antisense RNA interference. We then couple protein roadblock and TI to design minimal genetic NAND and NOR logic gates. Together, these results show the importance of processivity control for strong TI and demonstrate TI's potential for synthetic biology.