Functional and biophysical mapping of archaeal transcription complexes

基因表达的第一步是DNA转录成RNA，这是由RNA聚合酶（rnap）促进的。rnap是由12个亚基组成的大型酶。我们目前并不了解所有亚单位的功能，但对它们的许多功能有很好的工作知识。rnap与它们的模板（DNA）、底物（NTP）和产物（RNA）结合，并与调控转录因子相互作用。许多关于rnap的研究都是基于确定酶的结构。像RNAP这样的大型酶是动态的和灵活的，它们通常由一个刚性框架和可移动的部分组成，这些部分可以重新定向以适应与DNA/RNA和调节因子等相互作用。这种灵活性可能是RNAP功能的潜在机械基础。然而，即使预测到许多结构变化，也不可能直接观察到它们。我们的实验室已经开发了从单个亚基构建RNAP的方法。这些可以用荧光探针标记，荧光探针是分子信标，可以测量它们之间的距离和距离的变化。我们希望将复杂的荧光技术与我们的系统相结合，首次表征RNAP的动力学。

The first step in gene expression is transcription of DNA into RNA and this is facilitated by RNA polymerases (RNAPs). RNAPs are large enzymes that are made of twelve subunits. We do currently not understand the function of all subunits but have a good working knowledge of many of their functions. RNAPs bind to their template (DNA), substrate (NTP) and product (RNA) and they interact with regulatory transcription factors. Much research on RNAPs is based on determining the structure of the enzyme. Large enzymes such as RNAP are dynamic and flexible, they often consist of a rigid frame and movable parts that can reorient themselves to accommodate interactions with e. g. DNA/RNA and regulatory factors. This flexibility is likely to be the underlying mechanical basis of RNAP function. However, even though many structural changes have been predicted it has not been possible to observe them directly. Our laboratory has developed methods to build RNAP from individual subunits. These can be labelled with fluorescent probes - molecular beacons that allow to measure distances and moreover changes in distances between them. We want to combine sophisticated fluorescence technology with our system to characterise for the first time the dynamics of RNAP.