Development of advanced single-molecule fluorescence imaging methods to visualize coupled conformational changes in transcription

生物分子机器的可移动元件之间以及不同分子机器之间的耦合是生物机器功能和大多数生物过程调节的关键。然而，捕获这种蛋白质机器的瞬态耦合动力学很难在单分子水平上执行，主要是由于缺乏直接观察耦合分子运动的可靠方法。该项目专注于开发单分子荧光方法，利用基于单分子光谱的多色Foerster共振能量转移来捕获蛋白质与核酸相互作用中的耦合构象转变。新方法将应用于RNA聚合酶（负责将DNA复制成信使RNA的机器）模块的运动和RNA聚合酶处理的DNA分子。这种新方法及其在转录起始中的应用将使我们对基因表达的基本步骤有更深入的了解，并为靶向RNA聚合酶的新抗生素的产生提供新的机会；此外，这项技术创新应该适用于其他生物分子复合物和过程，因此应该引起显微镜开发人员和分子生物学家的兴趣。

Coupling between mobile elements of a biological molecular machine, as well as between different molecular machines, is key for the function of biological machinery and for the regulation of most biological processes. However, capturing the transient coupled dynamics of such protein machines is difficult to perform at the single-molecule level, mainly due to the absence of robust methodology for the direct observation of coupled molecular motions. This project focuses on developing single-molecule fluorescence methodology that utilizes multi-colour Foerster resonance energy transfer based on single-molecule spectra to capture coupled conformational transitions in protein interactions with nucleic acids. The new methods will be applied to the motions of modules of RNA polymerase (the machine responsible for copy DNA into messenger RNA) and the DNA molecules processed by RNA polymerase. This new methodology and its application to transcription initiation should provide a much more thorough understanding of fundamental steps in gene expression and offer new opportunities for the generation of new antibiotics targeting the RNA polymerase; further, the technological innovation should be applicable to other biomolecular complexes and processes, and thus should be of interest to the communities of microscopy developers and molecular biologists.