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

项目摘要 G-qudruplex（G4）是一种非典型的二级结构，可以在DNA和RNA中形成。人类基因组 含有超过400，000个潜在的G4形成序列（PQS），它们高度富集在 癌基因启动子和调控基因，强烈表明开关样功能与程序定位。 PQS在e中也很普遍。大肠杆菌基因组，位于重要的调控区域。事实上，许多研究 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，在转录过程中，G4形成序列位于非- 模板链导致R环（mRNA退火至模板链）的稳健形成，这反过来诱导 非模板链中的G4结构。值得注意的是，这种R环/G4结构驱动增强的转录 通过涉及R环的连续形成和释放的机制。我们表明，当定位在一个 质粒，即在扭转约束下，这种G4/R环结构可以上调或下调转录活性 这取决于与转录起始位点的距离。此外，携带5 'UTR-G4的mRNA（RG 4）导致 到在无细胞系统和大肠杆菌细胞中超过10倍增强的翻译。在测试了几个合理的 假设，我们提出RG 4结构通过阻止核糖体从核糖体上滑落来促进翻译。 mRNA。在这些令人兴奋的新发现的基础上，我们建议研究G4，R-loop和 在转录和翻译过程中存在超螺旋现象，主要表现在T7 RNAP系统和e. coli细胞。通过组合 定量生化工具和新开发的单分子平台， 在线性或质粒DNA的转录进展，我们将研究如何不同的序列，长度和 G4形成序列的位置导致R环形成、mRNA输出和蛋白质产生。完成 提出的目标将揭示G4、R环和超螺旋如何调节的结构-功能关系 转录和翻译活性。

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.