Functional and biophysical mapping of archaeal transcription complexes

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.

This proposal aims at investigating the molecular mechanisms of transcription and in particular RNA polymerase. Our previous studies have combined structural and functional data to investigate the mechanisms of transcription. Little is known about the conformational transitions of transcription complexes during the transcription cycle in solution. This is important since crystallographic information only represents snapshots of dynamic multistep processes. Using fluorescence technology including Förster Resonance Energy Transfer (FRET, both ensemble and single molecule level) and fluorescence anisotropy combined with a recombinant RNAP we want to investigate the conformational changes that are underlying the transcription process. In addition we want to apply Electron Spin Resonance (ESR) to map molecular structure by the introduction of nitroxide spin labels and double electron electron resonance (DEER). We want to produce labelled RNAP subunits, transcription factors and nucleic acid scaffolds to assess and analyse the formation and stability of transcription complexes and conformational changes within RNAP due to interactions with nucleic acids and basal factors (TBP, TFB, TFE). We will initially focus on the small RNAP subunits H, K, F and E and to the large subunits A' and B' subunits. Predicted conformational changes include a closure of the RNAP jaw domain (subunit A' and H), movement of the stalk module (F/E) and a closure of the RNAP clamp (A') domain. Together with our excellent collaborators in the UK, Germany and USA, we will apply a multidisciplinary approach by using Biochemistry, Molecular Biology and Biophysics.

Importance. Foremost our research aims at expanding the knowledge base of gene expression and transcription. We are working on RNA polymerase, a formidable molecular motor and the primary driver of gene expression - a molecule that by many is considered one of the top five most important enzymes in biology (next to the ribosome, DNA polymerases, Chaperonins and Proteasome). All life forms depend on it and it deserves our special attention. We have over the recent years made good progress with generating structural information and developing assays to characterise the catalytic activities of RNAP, however we do know very little about the dynamics of transcription, both in terms of conformational flexibility within, and dynamic assembly and disassembly of RNAP and RNAP-containing transcription complexes. The current research proposal aims at filling this gap in our understanding of RNAP. With the recent advances in our ability to manipulate recombinant systems and a sophisticated biophysical toolbox we now have the unprecedented opportunity for a comprehensive description of the molecular mechanisms of transcription. Furthermore, the overall conclusions drawn from the work are likely to be valid not only for RNAPs, but for molecular motors in general. Impact in the field. The insights gained from the proposed work is likely to have a high impact in the field of transcription because it attempts to establish a missing link in our understanding of the molecular mechanisms of RNAPs - a direct characterisation of the dynamic nature of transcription complexes. It has until recently not been possible to carry out this type of work on eukaryote-like RNAPs due to the lack of recombinant RNAP systems. To our knowledge no other laboratories are currently pursuing these highly ambitious objectives and this proposal therefore represent a unique opportunity for the BBSRC to support leading edge science and technological innovation. The results obtained from this work are also likely to make significant contributions to the scientific communities focused on the structure and function of complex enzymes, the regulation of gene expression and the molecular evolution in the three domains of life. In the long term is has the potential to aid antibacterial and anticancer drug development. Biomedical impact. Bacterial infections are a major cause of human and animal morbidity and mortality. RNAPs are important targets for antibiotics, because (i) RNAP activity is strictly required for bacterial cell growth and (ii) drugs are highly selective and can discriminate between bacterial and eukaryotic enzymes. Rifamycins are first line drugs for the treatment of tuberculosis, which by the WHO is estimated to have killed 1.7 million people in 2006. However, due to the occurrence of multi and extreme drug resistant pathogen strains there is currently an urgent need to develop improved inhibitors, such as the alpha-pyrones including myxopyronin. 3D structures cannot alone provide the information that is required to drive the research and development of novel drugs. Recombinant transcription systems, however, can play an important role in this process by illuminating structure-function relationships of RNAPs that are needed to rationalise the mechanisms of drug action. E. g. myxopyronin acts via a novel mechanism by interfering with the conformational flexibility of RNAP that is essential for transcription initiation. This project proposal aims at characterising conformational changes that are instrumental for RNAP function, and our results might contribute to drug development in the future. In summary, the understanding of the mechanisms of transcription and RNAP is important for a long-term biomedical strategy and has the potential to improve human health and quality of life.