Army Research Laboratory - West Campus, California Institute of Technology, 1200 East California Blvd, Pasadena, CA, 91125, USA.
US Army Combat Capabilities Development Command Chemical Biological Center, 8198 Blackhawk Rd, APG, MD, 21010, USA.
Arch Biochem Biophys. 2019 Oct 15;674:108045. doi: 10.1016/j.abb.2019.07.010. Epub 2019 Jul 19.
The T7 bacteriophage RNA polymerase (T7 RNAP) serves as a model for understanding RNA synthesis, as a tool for protein expression, and as an actuator for synthetic gene circuit design in bacterial cells and cell-free extract. T7 RNAP is an attractive tool for orthogonal protein expression in bacteria owing to its compact single subunit structure and orthogonal promoter specificity. Understanding the mechanisms underlying T7 RNAP regulation is important to the design of engineered T7-based transcription factors, which can be used in gene circuit design. To explore regulatory mechanisms for T7 RNAP-driven expression, we developed a rapid and cost-effective method to characterize engineered T7-based transcription factors using cell-free protein synthesis and an acoustic liquid handler. Using this method, we investigated the effects of the tetracycline operator's proximity to the T7 promoter on the regulation of T7 RNAP-driven expression. Our results reveal a mechanism for regulation that functions by interfering with the transition of T7 RNAP from initiation to elongation and validates the use of the method described here to engineer future T7-based transcription factors.
T7 噬菌体 RNA 聚合酶（T7 RNAP）是理解 RNA 合成的模型，是蛋白质表达的工具，也是细菌细胞和无细胞提取物中合成基因电路设计的执行器。由于其紧凑的单一亚基结构和正交启动子特异性，T7 RNAP 是细菌中正交蛋白质表达的有吸引力的工具。了解 T7 RNAP 调控的机制对于设计基于 T7 的工程转录因子很重要，这些转录因子可用于基因电路设计。为了探索 T7 RNAP 驱动表达的调控机制，我们开发了一种快速且具有成本效益的方法，使用无细胞蛋白质合成和声学液体处理仪来表征工程化的基于 T7 的转录因子。使用这种方法，我们研究了四环素操纵子与 T7 启动子的接近程度对 T7 RNAP 驱动表达的调控的影响。我们的结果揭示了一种通过干扰 T7 RNAP 从起始到延伸的转变来发挥作用的调控机制，并验证了这里描述的方法可用于工程化未来的基于 T7 的转录因子。
