Antisense transcription as a tool to tune gene expression

A surprise that has emerged from transcriptomics is the prevalence of genomic antisense transcription, which occurs counter to gene orientation. While frequent, the roles of antisense transcription in regulation are poorly understood. We built a synthetic system in Escherichia coli to study how antisense transcription can change the expression of a gene and tune the response characteristics of a regulatory circuit. We developed a new genetic part that consists of a unidirectional terminator followed by a constitutive antisense promoter and demonstrate that this part represses gene expression proportionally to the antisense promoter strength. Chip‐based oligo synthesis was applied to build a large library of 5,668 terminator–promoter combinations that was used to control the expression of three repressors (PhlF, SrpR, and TarA) in a simple genetic circuit (NOT gate). Using the library, we demonstrate that antisense promoters can be used to tune the threshold of a regulatory circuit without impacting other properties of its response function. Finally, we determined the relative contributions of antisense RNA and transcriptional interference to repressing gene expression and introduce a biophysical model to capture the impact of RNA polymerase collisions on gene repression. This work quantifies the role of antisense transcription in regulatory networks and introduces a new mode to control gene expression that has been previously overlooked in genetic engineering. Synopsis A synthetic system developed in Escherichia coli and a computational model provide mechanistic insights into antisense transcription and show that it is a reliable means of controlling gene expression and tuning regulatory circuits. A large library of terminator–promoter combinations is used to quantify the role of antisense transcription in regulatory networks. The degree to which antisense promoters repress gene expression is proportional to the strength of the antisense promoter. Antisense RNA and transcriptional interference each contribute equally to the total repression that can be achieved using antisense promoters. A set of unidirectional terminators and constitutive promoters can be combined in a modular manner to implement gene expression control. Graphical Abstract A synthetic system developed in Escherichia coli and a computational model provide mechanistic insights into antisense transcription and show that it is a reliable means of controlling gene expression and tuning regulat