A Methylation-Directed, Synthetic Pap Switch Based on Self-Complementary Regulatory DNA Reconstituted in an All E. coli Cell-Free Expression System,ACS Synthetic Biology

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A Methylation-Directed, Synthetic Pap Switch Based on Self-Complementary Regulatory DNA Reconstituted in an All E. coli Cell-Free Expression System
ACS Synthetic Biology ( IF 3.7 ) Pub Date : 2021-09-22 , DOI: 10.1021/acssynbio.1c00326
Emanuel G Worst ₁ , Marc Finkler ₁ , Marc Schenkelberger ₁ , Ömer Kurt ₁ , Volkhard Helms ₂ , Vincent Noireaux ₃ , Albrecht Ott ₁

Affiliation

Pyelonephritis-associated pili (pap) enable migration of the uropathogenic Escherichia coli strain (UPEC) through the urinary tract. UPEC can switch between a stable ‘ON phase’ where the corresponding pap genes are expressed and a stable ‘OFF phase’ where their transcription is repressed. Hereditary DNA methylation of either one of two GATC motives within the regulatory region stabilizes the respective phase over many generations. The underlying molecular mechanism is only partly understood. Previous investigations suggest that in vivo phase-variation stability results from cooperative action of the transcriptional regulators Lrp and PapI. Here, we use an E. coli cell-free expression system to study molecular functions of the pap regulatory region based on a specially designed, synthetic construct flanked by two reporter genes encoding fluorescent proteins for simple readout. On the basis of our observations we suggest that besides Lrp, the conformation of the self-complementary regulatory DNA plays a strong role in the regulation of phase-variation. Our work not only contributes to better understand the phase variation mechanism, but it represents a successful start for mimicking stable, hereditary, and strong expression control based on methylation. The conformation of the regulatory DNA corresponds to a Holliday junction. Gene expression must be expected to respond if opposite arms of the junction are drawn outward.

中文翻译：

基于在全大肠杆菌无细胞表达系统中重组的自我互补调控 DNA 的甲基化导向合成 Pap 开关

肾盂肾炎相关菌毛 (pap) 使尿路致病性大肠杆菌菌株 (UPEC) 能够通过泌尿道迁移。UPEC 可以在表达相应pap基因的稳定“开启阶段”和抑制其转录的稳定“关闭阶段”之间切换。调节区域内两个 GATC 动机之一的遗传性 DNA 甲基化在许多代中稳定了各自的阶段。潜在的分子机制只是部分理解。先前的研究表明，体内相变稳定性是转录调节因子 Lrp 和 PapI 的协同作用的结果。在这里，我们使用大肠杆菌无细胞表达系统，用于研究 pap 调节区的分子功能，该系统基于一种特殊设计的合成结构，两侧是两个编码荧光蛋白的报告基因，用于简单读出。根据我们的观察，我们认为除了 Lrp 之外，自互补调节 DNA 的构象在相位变化的调节中起着重要作用。我们的工作不仅有助于更好地理解相位变化机制，而且代表了模拟基于甲基化的稳定、遗传和强表达控制的成功开始。调节 DNA 的构象对应于 Holliday 结。如果连接处的相对臂向外拉，则必须预期基因表达会做出反应。

更新日期：2021-10-15

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