Engineering ligand-responsive RNA controllers in yeast through the assembly of RNase III tuning modules
Engineering ligand-responsive RNA controllers in yeast through the assembly of RNase III tuning modules
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DOI：
10.1093/nar/gkr090
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发表时间：
2011-07-01
影响因子：
14.9
通讯作者：
Smolke, Christina D.
中科院分区：
文献类型：
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作者：
Babiskin, Andrew H.;Smolke, Christina D.
细胞网络的编程以实现新的生物功能依赖于将分子信号的存在与基因调节活动联系起来的遗传工具的开发。最近，一组工程RNA控制器被描述，它能够通过RNaseIII酶Rnt1p直接切割转录本来调节酵母中的基因表达。在这里，我们描述了一种建立新型RNA传感驱动装置的策略，该策略基于将RNA适配子直接整合到Rnt1p发夹的一个区域中，从而调节Rnt1p的切割速率。我们证明了配体与整合适体结构域的结合与足以抑制Rnt1p加工的结构变化有关。通过在RNT1P开关平台中加入不同的功能模块，提出了三种优化开关动力学和配体响应性的调节策略。我们进一步证明，这些调谐模块可以以可预测的方式组合实施，以进一步改善交换机的调节响应特性。Rnt1p开关平台的模块化和可调性将允许快速优化和定制这一基因控制装置，从而为设计酵母复杂的遗传网络提供有用的工具。
The programming of cellular networks to achieve new biological functions depends on the development of genetic tools that link the presence of a molecular signal to gene-regulatory activity. Recently, a set of engineered RNA controllers was described that enabled predictable tuning of gene expression in the yeast Saccharomyces cerevisiae through directed cleavage of transcripts by an RNase III enzyme, Rnt1p. Here, we describe a strategy for building a new class of RNA sensing-actuation devices based on direct integration of RNA aptamers into a region of the Rnt1p hairpin that modulates Rnt1p cleavage rates. We demonstrate that ligand binding to the integrated aptamer domain is associated with a structural change sufficient to inhibit Rnt1p processing. Three tuning strategies based on the incorporation of different functional modules into the Rnt1p switch platform were demonstrated to optimize switch dynamics and ligand responsiveness. We further demonstrated that these tuning modules can be implemented combinatorially in a predictable manner to further improve the regulatory response properties of the switch. The modularity and tunability of the Rnt1p switch platform will allow for rapid optimization and tailoring of this gene control device, thus providing a useful tool for the design of complex genetic networks in yeast.