title: Antisense transcription as a tool to tune gene expression.
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.
authors.affiliation: Synthetic Biology Center, Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA.
authors.forename: Jennifer A N
authors.initials: JA
authors.lastname: Brophy
authors.affiliation: Synthetic Biology Center, Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA cavoigt@gmail.com.
authors.forename: Christopher A
authors.initials: CA
authors.lastname: Voigt
mesh_terms: D055592:Biophysical Phenomena; D012321:DNA-Directed RNA Polymerases / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D053263:Gene Regulatory Networks / Q000235:genetics*; D005818:Genetic Engineering; D011401:Promoter Regions, Genetic; D016372:RNA, Antisense / Q000235:genetics*; D058615:Synthetic Biology; D014158:Transcription, Genetic
chemical_list: D016372:RNA, Antisense; D012321:DNA-Directed RNA Polymerases
keywords: antisense transcription; design automation; genetic circuits; synthetic biology; systems biology