Molecular Mechanisms of Sigma Factor Inhibition in a Gene Expression Switch

We propose to study the way cells can completely transform their identity by activating a 'gene expression switch'. Such transformations are important to naturally maintain health e.g. in embryo development which begins with identical cells which then crucially change into the different types of cells that make up a developed human. Conversely, these transformations can cause harm e.g. when cells become cancerous or pathogens invade hosts. A gene expression switch involves a distinct programme of genetic instruction being deactivated and replaced with an alternative that leads to radical transformation of the cell's nature.

In this study my group will examine a particular gene expression switch event in bacterial spore formation which is partially responsible for the persistence of 'hospital superbugs' as spores are a long-lived bacterial form, resistant to cleaning agents and thriving in patients depleted of natural gut microflora.

In a gene expression switch there are specific proteins that choreograph genetic instruction in a highly regulated process. We intend to uncover the detailed molecular shapes of some of these proteins using indirect techniques as they are too small to see even using powerful microscopes. We specialise in measuring protein shapes and the way they fit together by producing them artificially in large quantities, with the help of bacteria which act as our 'protein factories'. We then deduce the proteins' molecular structures by processing their behaviour when we bounce X-rays off them or put them in strong magnetic fields. Each of these techniques has its strengths and weaknesses but our combined approach can yield complementary information filling in the gaps left by using just one of the methods. Collaborating with a microbiologist we will feed information into each others' experiments to build up a mechanistic picture of this gene expression switch in bacterial spore formation. For example, if we identify a mutation in one of our proteins that makes it bind more tightly to its partner our collaborator can make the same mutation within bacteria to test whether it has the predicted effect in living systems.

By solving this jigsaw puzzle we hope to be in a stronger position to design novel antibiotics to attack the increasing problem of bacterial drug resistance and the project also has longer term implications for understanding gene expression switches in all aspects of health and disease.

Both prokaryotic and eukaryotic cells can completely change their phenotypes by synchronising the shut-down of one gene expression programme with the activation of another. These highly choreographed events occur in numerous aspects of biology and are crucial for health and disease. Despite their critical importance much remains to be discovered about gene expression switches, especially at the level of three-dimensional macromolecular detail.
In B. subtilis sporulation a gene expression switch occurs in which the forespore shuts off its sigma factor F (SigF) driven programme of transcription to activate the genes controlled by sigma factor G (SigG). This switch is regulated by various mechanisms including a vital role for SigG inhibitor, Gin, and its newly discovered SigF inhibitor relative, Fin, a conserved sporulation protein about which little is so far known. Effecting this switch requires precise mechanisms to keep the new gene expression array in check until the old one is deactivated and, by extrapolation, mechanisms to maintain repression of the old programme once the new one is in play. This work will use a wide range of biophysical techniques to determine the molecular mechanisms of this important gene expression switch event. The structures of Fin, Gin and their interacting proteins and regulatory DNA sequences will be solved using X-ray crystallography and NMR and characterised by ITC and MST. Our results will inform in vivo mutagenesis studies in B. subtilis by our collaborator which, in turn, will feed back into our work to probe this mechanism in atomic level detail. With the ongoing problem of 'hospital superbugs' this detailed exploration of sporulation has the long-term potential to identify entirely novel approaches to therapeutic intervention and the development of new antibiotics.

The proposed research will have shorter and longer-term impact on Society and the economy:

SHORTER-TERM IMPACT: My move to the chemistry department at King's has already begun to generate new experimental ideas through combining interdisciplinary expertise with local colleagues. This study will thus have impact on research methods in the chemistry community as discussed in my 'academic beneficiaries' section and there will be knock-on effects from any discovery resulting from use of these techniques by other groups. I have a longstanding commitment to Public Engagement, described in more detail in my Pathways to Impact statement and I have plans to discuss my findings in non-scientific settings through the written and spoken word creating social impact by making the public more kindly disposed towards science while feeding back their ideas into the creativity of scientific planning. Since I am now at a University that teaches Arts subjects as well as science there is also far more opportunity for creating radically interdisciplinary impact. I have requested a 10K public engagement budget within this proposal to fund a pilot art project with Lavender Hill Studios which will lead to an immersive public exhibition that will directly engage a large number of people with diverse backgrounds and education.

LONGER-TERM IMPACT: The more information we have, on the detailed workings of the healthy and diseased body, the stronger is our potential to combat disease and promote health and wellbeing in the population. In this research programme my group will examine a gene expression switch event in B. subtilis in three-dimensional molecular detail. The resulting contribution will be significant in addressing the central biological question of how cells change their identity. This underpins developmental diseases and cancer in which inappropriate phenotypic changes cause serious harm. As an ideal goal, a molecular-level understanding of these processes supports the prospect of cellular 'reprogramming' strategies for therapeutic intervention in cancer and other illnesses. Such changes in gene expression are also responsible for developing resistance to drugs in cancer cells, anti-viral drug resistance and bacterial resistance to antibiotics; thus, a full three-dimensional understanding of gene expression switches will prove valuable in addressing some of the major challenges in modern medicine. These studies have the potential to identify novel antibiotic targets for gram-positive spore-forming pathogens such as Clostridium botulinum, Bacillus anthracis and Clostridium difficile, the source of nosocomial diarrhoea, pseudomembranous colitis and toxic megacolon in long-stay hospital patients. This would have enormous impact on society by addressing a uniquely modern problem that is getting worse. Moreover, from a synthetic biology perspective this work potentially offers scope for creating novel chimeric sigma factors that direct transcription of unique promoters which has the potential to add to the B. subtilis tractability toolkit which would have industrial, economic and academic impact.