Department of Chemistry and Konstanz Research School Chemical Biology, University of Konstanz, Konstanz, Germany.
RNA Biol. 2013 Jun;10(6):1010-6. doi: 10.4161/rna.24482. Epub 2013 Apr 1.
Synthetic biology approaches often combine natural building blocks to generate new cellular activities. Here, we make use of two RNA elements to design a regulatory device with novel functionality. The system is based on a hammerhead ribozyme (HHR) that cleaves itself to generate a liberated ribosome-binding site and, thus, permits expression of a downstream gene. We connected a temperature-responsive RNA hairpin to the HHR and, thus, generated a temperature-controlled ribozyme that we call thermozyme. Specifically, a Salmonella RNA thermometer (RNAT) known to modulate small heat shock gene expression by temperature-controlled base-pairing and melting was fused to the ribozyme. Following an in vivo screening approach, we isolated two functional thermozymes. In vivo expression studies and in vitro structure probing experiments support a mechanism in which rising temperatures melt the thermometer structure impairing the self-cleavage reaction of the ribozyme. Since RNA cleavage is necessary to liberate the RBS, these engineered thermozymes shut off gene expression in response to a temperature increase and, thus, act in a reverse manner as the natural RNAT. Our results clearly emphasize the highly modular nature and biotechnological potential of ribozyme-based RNA thermometers.
合成生物学方法通常结合天然构建块来产生新的细胞活动。在这里，我们利用两个 RNA 元件设计了一个具有新功能的调控装置。该系统基于锤头核酶 (HHR)，它自身切割以产生一个游离的核糖体结合位点，从而允许下游基因的表达。我们将温度响应性 RNA 发夹连接到 HHR 上，从而产生了一种我们称之为热酶的温度控制核酶。具体来说，将一种已知通过温度控制碱基配对和熔解来调节小热休克基因表达的沙门氏菌 RNA 温度计 (RNAT) 融合到核酶上。通过体内筛选方法，我们分离出两种功能齐全的热酶。体内表达研究和体外结构探测实验支持这样一种机制，即温度升高会使温度计结构融化，从而损害核酶的自我切割反应。由于 RNA 切割对于释放 RBS 是必要的，因此这些工程化的热酶会响应温度升高而关闭基因表达，因此它们的作用与天然 RNAT 相反。我们的结果清楚地强调了基于核酶的 RNA 温度计的高度模块化性质和生物技术潜力。
