Antisense transcription as a tool to tune gene expression.
Antisense transcription as a tool to tune gene expression.
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DOI：
10.15252/msb.20156540
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发表时间：
2016-01-14
影响因子：
9.9
通讯作者：
Voigt CA
中科院分区：
文献类型：
--
作者：
Brophy JA;Voigt CA
转录组学中出现的一个令人惊讶的现象是基因组反义转录的普遍存在，这种转录与基因的方向相反。虽然经常，反义转录在调控中的作用知之甚少。我们在大肠杆菌中建立了一个合成系统，以研究反义转录如何改变基因的表达并调节调控回路的响应特性。我们开发了一种新的遗传部分，由一个单向终止子，然后由一个组成型反义启动子，并证明这部分抑制基因表达的反义启动子强度成比例。应用基于芯片的寡核苷酸合成来构建5，668个终止子-启动子组合的大型文库，其用于在简单的遗传电路（非门）中控制三种阻遏物（PhlF、PXR和塔拉）的表达。使用该文库，我们证明了反义启动子可以用于调节调节电路的阈值，而不影响其响应功能的其他特性。最后，我们确定了反义RNA和转录干扰抑制基因表达的相对贡献，并引入了一个生物物理模型来捕获RNA聚合酶碰撞对基因抑制的影响。这项工作量化了反义转录在调控网络中的作用，并引入了一种新的模式来控制基因表达，这种模式以前在基因工程中被忽视。
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.