Abstract
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.
Highlights
Genetic engineers typically follow a simple scheme for controlling gene expression: a promoter and terminator flank the gene in the same orientation
We demonstrate that the antisense promoter represses gene expression in accordance with its strength and that the antisense transcription can cause a change in the threshold of inducible systems
A simple system was designed to quantify the impact of an antisense promoter on gene expression (Fig 1A)
Summary
Genetic engineers typically follow a simple scheme for controlling gene expression: a promoter and terminator flank the gene in the same orientation. Larger designs consisting of multiple genes are often organized where transcription is designed to proceed in one direction. In H. pylori, about half of the genes have at least one antisense promoter (Sharma et al, 2010) Some of this antisense transcription is the result of inefficient termination by intrinsic and rho-dependent terminators (Peters et al, 2012), it is often driven by promoters with well-defined regulatory motifs, such as housekeeping sigma factor binding sites (Dornenburg et al, 2010; Raghavan et al, 2012; Wade & Grainger, 2014). Antisense transcription can be constitutive or regulated under different environmental conditions (Beaume et al, 2010; Nicolas et al, 2012)
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