Deciphering molecular and kinetic bases of transcription with single molecule approaches

转录对细胞至关重要。像动态平衡、生长和分化这样的过程需要蛋白质数量的良好平衡，因此在时间上精确的转录调控是重要的。对于大多数基因来说，mRNA是在多个转录本的突发中产生的，然后是没有转录的非活跃期。已经观察到许多调节特性影响这种转录爆发，包括激活转录因子(TF)的结合，与共激活因子的相互作用，以及增强子-启动子的相互作用。然而，这些特征如何调控转录突发特性的机制和动力学细节，如突发频率、大小和持续时间，尚不清楚或缺失。通过这一提议，我们旨在了解转录调控的几个分子和动力学方面。我们计划使用人工TF和报告基因来操纵细胞内调节特性的机制和动力学特性，例如TF与其靶点结合的时间、近端增强子上的靶点数量或远端增强子和启动子之间的距离。我们将通过在活细胞和固定细胞中使用单分子荧光成像和单转录可视化来表征改变的性质，并监测辅助激活物相互作用和信使核糖核酸产生的变化。总体而言，我们的方法将对转录动力学的调节产生全面的定量洞察。

Transcription is vital to the cell. Processes such as homeostasis, growth and differentiation require well-balanced protein numbers, wherefore temporally precise regulation of transcription is important. For most genes, mRNA is produced in bursts of multiple transcripts, followed by inactive periods without transcription. Numerous regulatory traits have been observed to influence such transcriptional bursting, including binding of activating transcription factors (TFs), the interplay with co-activators, and enhancer-promoter interactions. However, the mechanistic and kinetic details of how these traits regulate properties of transcriptional bursts such as burst frequency, size and duration, are unclear or missing.With this proposal we aim at understanding several molecular and kinetic aspects of transcription regulation. We plan to manipulate mechanistic and kinetic properties of regulatory traits within the cell, such as the time a TF spends bound to its target site, the number of target sites at the proximal enhancer, or the distance between distal enhancer and promoter, using artificial TFs and reporter genes. We will characterize altered properties and monitor the changes to co-activator interaction and mRNA production by using single molecule fluorescence imaging and single transcript visualization in live and fixed cells. Overall, our approach will yield comprehensive quantitative insight into the regulation of transcription kinetics.