Molecular Mechanisms of Sigma Factor Inhibition in a Gene Expression Switch

Lead Research Organisation: King's College London
Department Name: Chemistry

Abstract

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.

Technical Summary

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.

Planned Impact

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.

Publications

10 25 50
 
Title Viewing the Invisible 
Description Kings College London and London Fine Art Studios have embarked upon an exciting project with the support of the BBSRC (Biotechnology and Biological Sciences Research Council). Viewing the Invisible brings together scientists and artists to explore the similarities in their working methods: Through the close study of shapes and constituent parts, scientists and artists alike, seek to demystify the human identity. This shared approach belies the popularly held belief that science and art reside at polar ends of a spectrum. In fact, it points to the ways in which they work together and can support each other to disseminate research and enrich understanding among a broader public. The outcome (detailed on the next page) is a multi-faceted display including short films, photographs, paintings and text, as well as accompanying activities, which we will display in a series of events around the country. We are holding a launch event in September 2019 at the National Portrait Gallery, potentially aligned with the theme of 'identity' in the Cindy Sherman exhibition. The 1.5 hour event will consist of a portrait painting demonstration from life, direct painting (alla prima) of a female fellow of the Royal Society (Dame Janet Thornton). The scientist will talk about her experimental methodology in conversation with the artist, Ann Witheridge, who will demonstrate the techniques and start of the painting from shapes, values and colour, explaining how in order to be a figuratively successful artist, one has to approach the subject abstractly, thinking of values and temperatures not eyes and mouths! Alongside the live-painting there will be a panel discussion featuring high-profile subjects from our project who will discuss the common ground between scientific and artistic approaches to exploring the underpinnings of identity. 
Type Of Art Artistic/Creative Exhibition 
Year Produced 2019 
Impact We haven't yet held our exhibition but it is planned for September 2019 in The Arcade in Bush House, a public facing venue on The Strand. We plan to evaluate our impact extensively and will report back in detail next year. 
 
Description We have now solved the molecular shapes of two different proteins (Fin and CsfB) that are responsible to changing normal bacteria into long-lived spores that survive in harsh conditions. We have many additional results that we are currently preparing for publication and by next we will report fully on all our objectives. Additionally, some experiments we have done in this grant led to some unexpected research questions about metabolic shutdown during sporulation - we have now secured a new BBSRC grant starting later this year which will address these.
Exploitation Route Our structural and functional data increase the current understanding of identity changes in cells which are important for many processes such as understanding embryo development, cancer and some hospital superbugs. Our structures might form the basis for rationally designed drugs.
Sectors Aerospace, Defence and Marine,Agriculture, Food and Drink,Chemicals,Education,Environment,Healthcare,Pharmaceuticals and Medical Biotechnology

 
Description One of our structures was adopted as 'featured structure' by the Protein Data Bank with an associated blog post giving some back story on how we solved it. As a result a software developer got in touch with us to use our data to improve the program that we used to calculate the structure. The post made a splash on social media, was retweeted for World Gin Day (as the protein is also known as Gin) and resulted in my being invited to speak at an art exhibition opening which gave rise to another collaboration and some important contacts.
First Year Of Impact 2018
Sector Creative Economy,Digital/Communication/Information Technologies (including Software),Education,Culture, Heritage, Museums and Collections
Impact Types Cultural,Societal

 
Description ITAS
Amount £3,000 (GBP)
Funding ID BB/R006091/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 05/2017 
End 06/2017
 
Description MicA, a novel protease adaptor in metabolic shutdown
Amount £441,343 (GBP)
Funding ID BB/S006877/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 06/2019 
End 05/2022
 
Description B. subtilis sporulation - structure/function collaboration with Prof. Amy Camp 
Organisation Mount Holyoke College
PI Contribution This is a research collaboration. We Skype every two weeks to update each other on research findings. From our end we solve structures and interactions of proteins using biophysics techniques.
Collaborator Contribution The Camp lab mostly perform in vivo microbiology work in Bacillus subtilis. In general they identify important proteins via genetic screens. We characterise these using structural and biophysical methods. Together we generate hypotheses which can then be tested in vivo by them and in vitro by us. Two people in my lab were able to spend time in the Camp lab thanks to BBSRC ITAS award and they have brought back important skills of working with B. subtilis such that we can now produce lysate important for experiments in our lab.
Impact We have published 2 papers together so far and generated two BBSRC project grants. We have many other papers in preparation. We have both spoken at European Spores Conference about our collaborative work.
Start Year 2015
 
Description Gin with a twist 
Form Of Engagement Activity Engagement focused website, blog or social media channel
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Media (as a channel to the public)
Results and Impact Our publication was featured on the front page of the PDB Europe website as 'featured structure' with an associated blog post.
Year(s) Of Engagement Activity 2018
URL http://www.ebi.ac.uk/pdbe/about/news/gin-twist
 
Description Interview on The Science Show 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact I was interviewed about my research by Robyn Williams for The Science Show which is broadcast to a large listening public by the Australian Broadcasting Corporation (ABC)
Year(s) Of Engagement Activity 2017
URL http://www.abc.net.au/radionational/programs/scienceshow/misshaped-proteins-the-cause-of-many-diseas...
 
Description K+ career speed-networking 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact I took part in a speed-networking careers event as part of K+ which is the KCL widening-participation programme for BAME and/or underprivileged local sixth-formers.
Year(s) Of Engagement Activity 2016
URL http://www.kcl.ac.uk/study/widening-participation/our-activities/k-plus/index.aspx
 
Description National Portrait Gallery Talk 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact For International Women's Day, I delivered one of the National Portrait Gallery's lunchtime lectures. These occur every other Thursday and are ticketed events open to the general public. I looked at the pioneering work of female structural biologists in the Gallery's Collection including Rosalind Franklin, Dorothy Hodgkin, Dame Louise Johnson, Dame Kathleen Lonsdale and Olga Kennard, explaining my science as I went along. There was a large audience and an interesting question and answer session afterwards.
Year(s) Of Engagement Activity 2017
URL http://www.npg.org.uk/whatson/event-root/march/lunchtime-lecture-02032017#.WK-LgvRBaWk.facebook
 
Description Olga Kennard interview 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Media (as a channel to the public)
Results and Impact I interviewed pioneering female X-ray crystallographer, Olga Kennard (who is 93) and wrote about it in Times Higher Education.
Year(s) Of Engagement Activity 2017
URL https://www.timeshighereducation.com/blog/international-womens-day-meeting-olga-kennard
 
Description PDB Europe Art Exhibition 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact I participated in a training event in which we planned projects with teachers to use protein structures in art classes.
Year(s) Of Engagement Activity 2018
 
Description STEM for Britain 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Policymakers/politicians
Results and Impact My Ph.D. student has competed with other Ph.D. students across the country to present her research in an accessible way to MPs. The vent will take place on 13th March and she has made a fantastic poster.
Year(s) Of Engagement Activity 2017
URL http://www.setforbritain.org.uk/2017event.asp
 
Description School Visit (Seven Kings, Essex) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact I visited Seven Kings School for their 'Women in Science' day and gave the same presentation three times to different groups of ~25 female sixth-formers about my career so far and work-life balance. I received positive feedback from students and teachers and my visit inspired the school to organise a trip to the HWB-NMR national facility in Birmingham.
Year(s) Of Engagement Activity 2016
 
Description Shadow a Scientist 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact My post-doc, Katherine Collins, and two Ph.D. students, Ewelina Krysztofinska and Nicola Evans, volunteered to introduce school age students to the lab with Scientist & co, Shadow a Scientist programme and also helped them with their CVs afterwards.
Year(s) Of Engagement Activity 2016
URL http://www.scientistsandco.org/projects/shadow-scientist/