Gene Expression Modulated by G4, R-loop and DNA Supercoiling

项目总结 G-qudraplex(G4)是一种非规范的二级结构，在DNA和RNA中都可以形成。人类基因组 包含超过400,000个潜在的G4形成序列(PQ)，它们在上游高度浓缩 癌基因启动子和调控基因，强烈表明具有程控定位的开关样功能。 PQS也广泛存在于大肠杆菌基因组中，位于重要的调控区。事实上，许多研究都有 证明了G4在上调或下调基因组过程中的作用，包括复制、转录和 翻译。我们最近的研究表明，在转录中，G4形成序列位于非 模板链导致R-环(退火到模板链的mRNA)的强健形成，这反过来又诱导 非模板链中的G4结构。值得注意的是，这种R-loop/G4结构推动了增强转录 通过一种涉及R-环的连续形成和释放的机制。我们展示了当定位在 质粒即在扭转约束下，这种G4/R环结构可以调节转录活性的上调或下调 取决于距转录起始点的距离。此外，5‘端非编码区G4携带的mRNA(RG4)导联 在无细胞系统和大肠杆菌细胞中将翻译能力提高10倍以上。在测试了几个看似合理的 假设，我们认为RG4结构通过阻止核糖体滑动来促进翻译 MRNA.在这些令人兴奋的新发现的基础上，我们建议调查G4、R-LOOP和 在转录和翻译中主要是在T7RNAP系统和在大肠杆菌细胞中发生超螺旋。通过组合 适合分步测量的定量生化工具和新开发的单分子平台 在线性或质粒DNA中转录的进展，我们将检查不同序列、长度和 G4形成序列的位置决定了R-环的形成、信使核糖核酸的合成和蛋白质的合成。完成 提出的目标将揭示G4、R环和超线圈如何调节的结构-功能关系 转录和翻译活动。

PROJECT SUMMARY G-qudruplex (G4) is a noncanonical secondary structure that can form in both DNA and RNA. Human genome contains over 400,000 potential G4 forming sequences (PQS) and they are highly enriched in upstream of oncogene promoters and regulatory genes, strongly suggesting a switch-like function with programed positioning. PQS is also prevalent in e. coli genome, located in important regulatory regions. Indeed, many studies have demonstrated the role of G4 in up or downregulating genomic processes including replication, transcription and translation. Our recent study demonstrated that in transcription, G4 forming sequence located in the non- template strand leads to a robust formation of R-loop (mRNA annealed to template strand), which in turn, induces G4 structure in the non-template strand. Remarkably, such R-loop/G4 structure drives enhanced transcription by a mechanism that involves successive formation and release of R-loop. We show that when positioned in a plasmid i.e under torsional constraint, such G4/R-loop structure can tune the transcription activity up or down depending on the distance from the transcription start site. Furthermore, 5’UTR-G4 bearing mRNA (RG4) leads to over 10-fold enhanced translation in a cell-free system and in e.coli cells. Upon testing several plausible hypotheses, we propose that the RG4 structure promotes translation by blocking ribosomes from sliding off the mRNA. Building on these exciting new findings, we propose to investigate the impact of G4, R-loop and supercoiling in transcription and translation primarily in T7 RNAP system and in e. coli cells. By combining quantitative biochemical tools and newly developed single molecule platforms suited to measure stepwise progression of transcription in a linear or plasmid DNA, we will examine how different sequence, length and position of G4 forming sequence leads to R-loop formation, mRNA output and protein production. Accomplishing the proposed goals will reveal the structure-function relationship of how G4, R-loop and supercoiling regulates transcription and translation activity.