Positive control of the primary sigma factor of actinomycetes

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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.