Spatio-termporal analysis of transcription dynamics in vivo

我们关于转录、其动力学和调控的大部分知识是通过体外生物化学和遗传学方法获得的，或者在真核细胞中引入合成基因后通过显微镜实验获得的。在这里，我们将使用先进的荧光显微镜方法，即超分辨率、单分子显微镜和三维单分子跟踪，在最小扰动系统中研究体内转录动力学。我们将利用一个标志性的生物系统，它已经被使用了几十年来研究mRNA生命周期的不同方面：触须摇蚊唾液腺细胞核内的大Balbiani环(BR)转录本。在这些细胞中，BR和热休克转录位点可以在视觉上很好地识别。此外，大量的转录过程在多线染色体内平行进行，这极大地提高了通过光学显微镜观察特定分子事件的机会。本项目的目标是在尽可能自然的条件下分析转录位点的拓扑结构、转录动力学及其在体内的调控。我们将集中讨论与体内转录的空间组织和动力学有关的一些基本问题：在一个自然系统中的转录速率是多少？差饷的分布情况如何？整个基因的速率是恒定的吗？转录在空间上是如何组织的？如何对转录进行调控？本研究的目的是在单分子水平上促进对体内转录机制的理解。

The most of our knowledge about transcription, its dynamics and regulation was acquired by biochemical and genetic approaches in vitro, or by microscopy experiments after introducing synthetic genes in eucarytotic cells. We will study here transcription dynamics in vivo in a minimally perturbed system using advanced fluorescence microscopy methods, i.e. super resolution, single molecule microscopy and 3-dimensional single‐molecule tracking. We will take advantage of an iconic biological system that has been used for decades to study diverse aspects of the mRNA life cycle: the large Balbiani ring (BR) transcripts within the nuclei of salivary gland cells of Chironomus tentans. In these cells the BR and heat shock transcription sites can visually well be identified. Furthermore, a high number of transcription processes takes place in parallel within the polytene chromosomes, what strongly improves the chance to observe specific molecular events by light microscopy. The goal of this project is to analyze the topology of the transcription sites, the dynamics of transcription and its regulation in vivo under conditions that are as native as possible. We will focus on a number of fundamental questions concerning the spatial organisation and dynamics of transcription in vivo: What is the transcription rate in a native system? What is the distribution of rates? Is the rate constant over the complete gene? How is transcription spatially organised? How can transcription be regulated? The purpose of the study is to advance the understanding of transcription mechanisms in vivo at the level of single molecules.