Positive control of the primary sigma factor of actinomycetes

Bacteria are of enormous economic and social importance and we benefit from their ability to synthesise valuable products (e.g. antibiotics, biofuels), preserve food, and bio-transform industrial waste, while facing a constant battle against pathogenic bacteria that evolve ways of evading current treatments. In order to fully exploit bacteria for industrial processes and to meet challenges faced by emerging pathogens we need to understand how bacteria control the expression of their genes. Gene expression starts with the binding of an enzyme called RNA polymerase to DNA promoter elements that are located upstream of protein coding sequences. RNA polymerase (RNAP) catalyses the production of an RNA copy of the gene in a process called transcription which, in most cases, is then translated by ribosomes in the production of proteins. The frequency of transcription initiation is controlled by regulatory proteins, most of which bind to DNA in the promoter region, often also interacting with RNAP. RNAP is a multi-subunit complex consisting of a core enzyme of five subunits and a dissociable sixth subunit called sigma that is required for promoter binding and the melting of DNA to reveal the template strand. Soon after transcription initiation, after RNAP has escaped from the promoter, the sigma subunit dissociates. Bacteria usually contain multiple sigma factors including one essential primary sigma factor responsible for most transcription in actively growing cells, and several alternative sigma factors with more specialised roles that reprogrammed RNAP to recognise new sets of promoters and switch on distinct groups of genes. We have discovered an unorthodox transcription factor called RbpA that binds to the primary sigma factor and stimulates transcription initiation. We do not understand how RbpA stimulates transcription, but it does not bind DNA and so its mechanism is distinct from standard DNA-binding activators. RbpA homologues are only found in the actinomycete family of bacteria, which includes bacteria of industrial and medical significance, most notably the Streptomyces and Mycobacteria genera. The Streptomyces genus is the source of most clinically-used antibiotics and many chemotherapeutic agents. On the other hand the Mycobacterium genus includes the most important global bacterial pathogen, M. tuberculosis, which infects a third of the world's population and kills approaching 2 million humans per year. This proposal aims to develop our understanding of transcription initiation in actinomycetes and has important implications for 1) how antibiotic biosynthetic genes are transcribed and 2) the development of new drugs to inhibit mycobacterial RNAP, the target of the front-line TB antibiotic rifampicin. Our finding that RbpA binds to the primary sigma factor suggests that it plays a major role in transcription initiation, which is consistent with a dramatic slowing of growth rate when the protein is absent from the model Streptomyces strain S. coelicolor. In this project we will investigate how RbpA influences the activity of sigma by monitoring the localisation of sigma on chromosomal DNA in its presence or in its absence. We will also determine the structure of RbpA alone and when bound to sigma, which will allow us to build a model for how RbpA works in the context of the larger RNAP complex and might reveal new ways to inhibit it. Finally we will combine the structural models with new genetic and biochemical approaches to understand how RbpA activates transcription. The outcomes of the project will have wide ranging implications for how transcription initiates in actinomycetes and will be of interest to other researchers that are interested in finding new ways to inhibit RNA polymerase.

Transcription initiation in bacteria depends on a multi-subunit catalytic core RNA polymerase in combination with a dissociable sigma subunit that directs RNA polymerase to promoter elements and provides a protein scaffold that stabilizes the non-template strand upon DNA melting. All bacteria contain a primary essential sigma factor that is responsible for most transcription in actively growing cells. In the actinomycetes, including Streptomyces coelicolor and Mycobacterium tuberculosis, a small protein, RbpA, binds to the primary sigma factor and stimulates its activity. RbpA is unorthodox in that it contains no obvious DNA binding determinants and interacts with conserved region 1.2-2. This multidisciplinary work programme will apply genome-wide, structural, genetic, and biochemical approaches to understand RbpA structure and function. Structural studies will be conducted with the M. tuberculosis SigA-RbpA pair, whereas the genetic and biochemical approaches will be carried out using S. coelicolor proteins. We will use ChiP-chip approaches to investigate the global influence of RbpA on sigma distribution and activity, which will also allow us to determine any influence on the dissociation of sigma from elongating transcription complexes. We will expand our preliminary NMR experiments to solve the solution structure of RbpA then solve the solution structure of RbpA bound to conserved region 1.2-2, which will allow us to build a model in the context of RNA polymerase holoenzyme. In order to relate structure to function we will conduct a series of genetic and biochemical experiments to understand how RbpA activates transcription. We will attempt to isolate suppressor mutants that no longer require RbpA for rapid growth and dominant negative rbpA mutants that might be active in sigma binding but defective in activation. In addition we will apply kinetic approaches to determine the step in the transcription initiation process at which RbpA acts

This is cutting edge interdisciplinary research, which has the potential for substantial economic impact. It will also maintain the supply of quality research expertise in actinomycetes in the UK whilst enabling and promoting knowledge transfer between academia and companies. The following beneficiaries have been identified (a), and methods of how they will benefit (b), and what will be done to ensure that they have the opportunity to benefit from this research (c), are detailed. Beneficiary One (a) Large pharma (e.g. Pfizer, Novartis, GSK) and smaller Biotech companies (e.g., Novacta, Biotica), or not-for-profit organisations (e.g. TB Alliance) that are screening or engineering actinomycetes for bioactive compounds, or are developing new antibiotics that inhibit RNA polymerase. (b) Outcomes might benefit the commercial sector in two ways: i) by enhancing our understanding of transcription initiation in antibiotic producers, new ways to enhance production or switch on cryptic biosynthetic genes might emerge; ii) a new structure-based understanding of transcription initiation in Mycobacterium tuberculosis, will benefit pharma in the development of novel RNA polymerase inhibitors or in the development of novel rifamycin derivatives. (c) This is basic blue-sky research so it is too early to identify industrial partners. Most importantly, IP will be protected to enhance value and maximise opportunities for collaborative research or licensing. Through Sussex Business and Enterprise we will target companies that we feel might benefit from any IP generated through Sussex IP. Microarray metadata will be freely available on-line through Arrayexpress and findings will be published in a timely fashion in peer-reviewed journals. Beneficiary Two (a) Members of the wider academic community investigating gene regulation including systems approaches to modelling bacterial networks. (b) The outcomes might also reveal new models for transcriptional control, thereby stimulating research in diverse systems. Large 'omic sets will be produced that will be valuable for mathematical modellers. (c) Primarily through publication, presentation at international research meetings and the development of new collaborations where appropriate. Beneficiary Three (a) Skills, training and knowledge economy (b) The PDRA and any undergraduate, postgraduate, or intern students that contribute to the project will be trained with key interdisciplinary skills that will be extremely valuable for UK industry and contribute to the knowledge economy and increase the economic competitiveness of the UK. (c) The PDRA (Sussex) and technician (Imperial) will be trained in key techniques and good laboratory practice by the investigators. Staff will be encouraged to be innovative. There are opportunities for training of undergraduate, postgraduate, or intern students at both Imperial and Sussex. For example, an MChem student has made significant input into the initial RbpA assignments at Imperial. Beneficiary Four (a) Public sector health professionals (b) Clinicians and senior health providers benefit from an improved understanding of where key medicines, such as antibiotics, come from and how they act. (c) The PI will contribute to local network groups including the Brighton and Sussex INFECTION AND IMMUNOLOGY Research Network (http://www.sussex.ac.uk/business/medical/immunology.php) - indeed the PI gave a talk on the subject of this proposal to this group in 2009. Beneficiary Five (a) International Development (b) In the long-term the outcomes might play a significant role in the development of novel treatments for TB, which infects more than a third of the world's population and is a scourge on the economy of developing nations. (c) No-profit organisations such as TB Alliance are pushing forward with the development of novel TB treatments. We will engage with specific arms of the TB Alliance (e.g. RNA polymerase inhibitors) when appropriate.