Probing temporal sequences and molecular mechanisms of transcription initiation by single molecule fluorescence microscopy in living eukaryotic cells

转录因子与其在DNA上的靶点的初始结合会触发一系列事件，最终导致转录机制的组装和RNA的产生。虽然对于许多基因来说，参与启动途径的分子物种是已知的，但关于它们相互作用以及与DNA相互作用的详细动力学信息在很大程度上是缺乏的。因此，关于转录起始的时间协调、从转录因子结合到转录起始的信息流以及这一过程的效率等关键问题仍然没有答案。这一建议旨在通过测量和直接操纵细胞动力学来理解基因转录的重要动力学方面及其启动。我们将使用单分子荧光显微镜技术，如反射光片显微镜，来监测参与转录启动的单个生物分子在活的哺乳动物细胞细胞核中的行为，并提取它们与相互作用伙伴相互作用的动力学信息。在项目的第一部分，我们计划开发在DNA上停留时间可控的人工转录因子，并测量基因转录的时间分布如何依赖于这一参数。在第二部分中，我们将研究基本转录因子的动态以及它们组装到转录机制中的途径。

The initial binding of a transcription factor to its target site on DNA triggers a series of events that finally lead to the assembly of the transcription machinery and the production of RNA. While for many genes the molecular species participating in the initiation pathway are known, detailed kinetic information on their interactions with one another and with DNA is largely missing. Thus, key questions concerning the temporal coordination of transcription initiation, the flow of information from transcription factor binding to the onset of transcription and the efficiency of this process remain unanswered. This proposal aims at understanding important dynamical aspects of gene transcription and its initiation by both measuring and directly manipulating cellular dynamics. We will use single molecule fluorescence microscopy techniques such as reflected light sheet microscopy to monitor the actions of individual biomolecules involved in transcription initiation in the nuclei of living mammalian cells and extract kinetic information on their interplay with interaction partners. In the first part of the project we plan to develop artificial transcription factors with a controllable residence time on DNA and measure how the temporal profile of gene transcription depends on this parameter. In the second part, we will study the dynamics of essential basal transcription factors and pathways of their assembly into the transcription machinery.