Kinetic analysis of aptazyme-regulated gene expression in a cell-free translation system: modeling of ligand-dependent and -independent expression.
Kinetic analysis of aptazyme-regulated gene expression in a cell-free translation system: modeling of ligand-dependent and -independent expression.
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无细胞翻译系统中适体酶调节的基因表达的动力学分析：配体依赖性和非依赖性表达的建模。
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发表时间：
2012
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通讯作者：
T. Yomo
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作者：
Shungo Kobori;N. Ichihashi;Y. Kazuta;Tomoaki Matsuura;T. Yomo
Aptazyme作为基于RNA的基因表达开关对几种类型的化合物做出反应是有用的。开关能力最重要的性质之一是信噪比(S/N)，即有配体存在的基因表达与没有配体的基因表达的比率。本研究旨在定量地了解端粒酶S/N的比值是如何由参与基因表达的因素决定的，这些因素包括转录、核糖核酸自切、核糖核酸降解、蛋白质翻译及其配体依赖性。在一个无细胞翻译系统中，我们使用两种不同性质的开关适配酶进行基因表达的切换，并构建了一个动力学模型，定量描述了参与切换的RNA和蛋白质物种的动力学。理论和实验分析都一致地证明，决定S/N比率绝对值和动力学的因素高度依赖于在没有配体的情况下翻译的途径：来自配体独立切割的RNA的翻译还是来自未切割的RNA的泄漏翻译。该模型可用于评估制约S/N比值的因素，从而更有效地改良端粒酶。
Aptazymes are useful as RNA-based switches of gene expression responsive to several types of compounds. One of the most important properties of the switching ability is the signal/noise (S/N) ratio, i.e., the ratio of gene expression in the presence of ligand to that in the absence of ligand. The present study was performed to gain a quantitative understanding of how the aptazyme S/N ratio is determined by factors involved in gene expression, such as transcription, RNA self-cleavage, RNA degradation, protein translation, and their ligand dependencies. We performed switching of gene expression using two on-switch aptazymes with different properties in a cell-free translation system, and constructed a kinetic model that quantitatively describes the dynamics of RNA and protein species involved in switching. Both theoretical and experimental analyses consistently demonstrated that factors determining both the absolute value and the dynamics of the S/N ratio are highly dependent on the routes of translation in the absence of ligand: translation from the ligand-independently cleaved RNA or leaky translation from the noncleaved RNA. The model obtained here is useful to assess the factors that restrict the S/N ratio and to improve aptazymes more efficiently.