Functional and biophysical mapping of archaeal transcription complexes

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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.