Biological functions that depend upon the bridge helix of RNA polymerase

Cells in all living organisms work through the concerted actions of tiny machines that carry out major transformations to allow growth and adaption. These tiny machines using the building blocks of life as their substrates, and form valuable materials from them, but also dismantle such materials when times so require.Ultimately, the DNA in cells is the blue print for these tiny machines, and will set how the machines interact with each other, the cells components, and importantly DNA itself. The genetic information in DNA must be accessed to allow cells to grow, adapt and to differentiate. Access is granted by the regulated activity of the RNA polymerase enzyme, a large and complex protein which copies DNA into an RNA template from which proteins are then produced. In this proposal we seek to work out how one conserved structural feature of RNA polymerase communicates with other conserved structural features of RNA polymerase to achieve 'full' functionality (in terms of accessing the information in DNA).The coupling interactions we plan to study-which can be thought of as being like the direct coupling of mechanical parts that allow engines to work and deliver force- are proposed to be required for the full functionality of RNA polymerase, but to date are not proven to exist. Specifically we will examine (i) interactions between the so called switch regions of RNA polymerase and one end of a long helical feature called the bridge helix; (ii) determine why altered forms of the bridge helix (eg the E. coli F773A mutant) works well in vitro but fails to support in vivo growth of cells;(iii) determine where points of bending in the alpha helical bridge helix are and what they are important for and (iv) look at how an RNA polymerase associated protein that directs it to specific DNA sequences (termed the promoter-specific sigma factor) can impact upon the functioning of the bridge helix. Gaining insights into how bacterial RNA polymerase works has important implications for the understanding of the functioning of all multi-subunit RNA polymerases, as well as in providing knowledge to help in the design of new anti-microbials that target the transcription apparatus.Our final aim is closely related to this longer term goal, since it seems that the feature of the RNA polymerase we wish to study is very flexible and may need to kink to work, indicating it might be captured in an unfavourable configuration by bespoke RNA polymerase-binding chemicals that would then act as inhibitors of its activity. We believe that through knowing how the RNA polymerase enzyme functions we can contribute to new strategies to mange infections and disease, as well as found new ways to improve biotechnological applications, such as protein and bio-chemicals productions.

Multisubunit RNA polymerases (RNAP) contain a major feature that is a long alpha helix, appears dynamic in nature and is proposed to make multiple interactions with features of the RNAP associated with a wide range of sub-functionalities - such as nucleic acid template binding and translocation,the nucleotide addition cycle (RNA synthesis), RNA cleavage and closure of RNAP around downstream DNA. We systematically conducted comprehensive alanine scanning mutagenesis of this helix in the biochemically and genetically tractable E. coli bacterial RNAP. We observed phenotypes consistent with bridge helix-switch region interactions; indicating closure of the RNAP around the DNA template downstream of the transcription start-site would depend on a network of interactions between the bridge helix and the switch regions. One aim of this proposal is to provide evidence for such a functional network by comparing the phenotypes of the bridge helix, switch region and double mutants to establish where common or parallel pathways operate for closure of RNAP. A second aim is to establish why one bridge helix variant functions well in all the in vitro assays conducted so far, but functions poorly in vivo; implying some factor-specific or promoter DNA or gene-specific interaction is defective in vivo - potentially revealing a hitherto unknown link to the functioning of RNAP supported by the bridge helix. The third aim is to identify the kinking regions of the BH, first proposed for a thermophilic archael RNAP. The fourth aim of the project is to establish which activities of the RNAP that are dependent upon the integrity of the bridge helix are influenced by the promoter-specific sigma factor,leading to testable proposals about the molecular pathways for communication between sigma factors and the bridge helix, which we envisage will provide new insights into the innate resistome of bacteria, where susceptibility of RNAP to certain antibiotics is relieved by sigma factors.

Our work is intended to probe inferences arising from available detailed structural snapshots of the transcription process, and so will help in validating derived models that seek to explain the functionality of multi-subunit RNA polymerases arising from many years of intensive and successful structural biology research. Basic research best contributes to social and economic goals when targeted to areas that can benefit from additional fundamental knowledge [Nelson, R.R., 2004. The market economy, and the scientific commons. Res. Policy 33, 455-471.Rosenberg, Nathan, and Richard R. Nelson (1994), 'American Universities and Technical Advance in Industry,' Research Policy 23: 323-348. Rosenberg, Nathan (1982), 'How Exogenous is Science?' in Inside the Black Box (NY: Cambridge University Press), p. 141-159]. Through studying the action of RNA polymerase, we expect to be able to accelerate the use of RNA polymerase variants and their associated control proteins in emerging synthetic biology research and application, as well as providing valuable insights into how RNA polymerase functioning might be corrupted by antimicrobials, of interest to the pharmaceutical industry and biotechnology firms. A number of structures exist within IC for exploitation of knowledge gained and the development of beneficial applications. For example, we could make use of the expertise offered by IC Innovations in the area of translating research into marketable products. In addition, we have the opportunity to benefit from input and advice from IC Drug Discovery Centre's multi-disciplinary team whose remit is to translate early research into drug discovery projects.The usual channels of knowledge transfer will communicate results to the industrial sector (i.e. peer-reviewed publications, international and national conferences etc.). The IC Business Development team would be a valuable resource in supporting any (long-term) future commercial development arising from this research. Similarly, this would benefit from the expertise offered by IC Innovations in the area of translating research into marketable products. The type of laboratory work planned will provide the named RA with skills in many post-genomic technologies and protein-nucleic acid biochemistry. The placement of masters students, undergraduate students and PhD students in our respective labs will help to expose the next generation of bio-science researchers to hypothesis generation and modern experimental execution. Through presentations at International and National meetings we will contribute to the knowledge base around one of the most important and fundamental cellular processes. Participation in the University outreach program and use of the outreach laboratory allows us to engage with school groups visiting Imperial College, and to show them something of our current work. For the proposed ChIP-chip work, we will work closely with Oxford Gene Technology (microarray analysis) and Prof. Michael Stumpf (statistical analysis of microarray data, and co-author of several papers with us). Links with both parties are readily available to us as part of current BBSRC funded projects. Other expertise relevant to the project will be provided by groups/facilities within IC, such as the Centre for Structural Biology (CSB) (e.g. for the structural modeling of the biochemical data) and the Centre for Bioinformatics (CBI) (e.g. for microarray data analysis in aim 2). Links to CSB and CBI are also readily available to us as part of current BBSRC funded projects. Interactions with these groups, mechanisms for interactions, sharing and archiving of data, facilities, reagents and expertise are already in place.