用于基因表达转录后调控的可调核糖调节开关
Tunable Riboregulator Switches for Post-transcriptional Control of Gene Expression.
作者信息
Krishnamurthy Malathy, Hennelly Scott P, Dale Taraka, Starkenburg Shawn R, Martí-Arbona Ricardo, Fox David T, Twary Scott N, Sanbonmatsu Karissa Y, Unkefer Clifford J
机构信息
Bioenergy and Biome Sciences, Bioscience Division, ‡Theoretical Biology and Biophysics, Theoretical Division, Los Alamos National Laboratory , Los Alamos, New Mexico 87545, United States.
出版信息
ACS Synth Biol. 2015 Dec 18;4(12):1326-34. doi: 10.1021/acssynbio.5b00041. Epub 2015 Jul 27.
Until recently, engineering strategies for altering gene expression have focused on transcription control using strong inducible promoters or one of several methods to knock down wasteful genes. Recently, synthetic riboregulators have been developed for translational regulation of gene expression. Here, we report a new modular synthetic riboregulator class that has the potential to finely tune protein expression and independently control the concentration of each enzyme in an engineered metabolic pathway. This development is important because the most straightforward approach to altering the flux through a particular metabolic step is to increase or decrease the concentration of the enzyme. Our design includes a cis-repressor at the 5' end of the mRNA that forms a stem-loop helix, occluding the ribosomal binding sequence and blocking translation. A trans-expressed activating-RNA frees the ribosomal-binding sequence, which turns on translation. The overall architecture of the riboregulators is designed using Watson-Crick base-pairing stability. We describe here a cis-repressor that can completely shut off translation of antibiotic-resistance reporters and a trans-activator that restores translation. We have established that it is possible to use these riboregulators to achieve translational control of gene expression over a wide dynamic range. We have also found that a targeting sequence can be modified to develop riboregulators that can, in principle, independently regulate translation of many genes. In a selection experiment, we demonstrated that by subtly altering the sequence of the trans-activator it is possible to alter the ratio of the repressed and activated states and to achieve intermediate translational control.
直到最近,改变基因表达的工程策略一直集中在使用强诱导型启动子进行转录控制,或者采用几种方法之一来敲除无用基因。最近,已开发出用于基因表达翻译调控的合成核糖调节因子。在此,我们报告了一种新型模块化合成核糖调节因子类别,它有可能精细调节蛋白质表达,并独立控制工程代谢途径中每种酶的浓度。这一进展很重要,因为改变特定代谢步骤通量的最直接方法是增加或降低酶的浓度。我们的设计在mRNA的5'端包含一个顺式阻遏物,它形成一个茎环螺旋,封闭核糖体结合序列并阻断翻译。一个反式表达的激活RNA释放核糖体结合序列,从而开启翻译。核糖调节因子的整体结构是利用沃森-克里克碱基对稳定性设计的。我们在此描述了一种能完全关闭抗生素抗性报告基因翻译的顺式阻遏物和一种能恢复翻译的反式激活剂。我们已经确定可以使用这些核糖调节因子在很宽的动态范围内实现基因表达的翻译控制。我们还发现可以对靶向序列进行修饰,以开发原则上能够独立调节许多基因翻译的核糖调节因子。在一项筛选实验中,我们证明通过微妙地改变反式激活剂的序列,有可能改变抑制态和激活态的比例,并实现中间水平的翻译控制。