Organisation and regulation of bacterial enhancer-binding proteins

Lead Research Organisation: Imperial College London
Department Name: Infectious Disease


RNA polymerase (RNAP) is a fundamental cellular machinery responsible for converting genetic information stored in DNA to another genetic molecule, called RNA, that can then be converted to protein or act in another regulatory or structural capacity. Accessing information in DNA occurs in a complex, highly controlled process called gene transcription and the core molecular machinery, the RNAP enzyme, is conserved from bacteria to humans. DNA is normally organised in chromosomes which organise DNA into higher order structures. Gene transcription is a highly regulated event in development and a major response to growth and environmental stimuli in all known living systems. Although significant advance has been made towards understanding how RNAP functions as an enzyme, including the work recognised by the Nobel Prize in Chemistry in 2006, how it is controlled by factors that signal special cellular states and events, is still poorly understood.

We are studying a unique system in bacteria that responds to bacterial stress and affects the ability of bacteria to respond to environmental changes, therefore affecting its ability to infect as a pathogen or propagate in a biotechnological setting. The key unique transcription factor, called sigma54, binds to RNAP and normally inhibits RNAP to prevent gene expression. Following a set of complex transactions with special control proteins that utilise the energy currency of the cell, a molecule called ATP, this system is then activated in a remodeling event to allow the RNAP to transcribe key genes in response to e.g. changes in the environment. These controlling activator proteins respond to a wide range of signals and are organised remotely on the DNA from RNAP. Therefore how these components are brought together to productively interact with each other and how the DNA is organised in this system as well as how signals regulate this system are extremely important to understand.

In this current proposed research, we plan to utilise the latest developments in life sciences technologies, especially using electron microscopy, to study these complex protein-DNA assemblies and how they change upon environmental signals to allow a regulated gene expression event. Such work is likely to shed light onto how RNAP in humans, plants and animals is activated. Furthermore, our approach of looking at large complex assemblies in transcription will bring us one step closer to studying these systems in the context of a complete chromosome and in intact cells. Furthermore, we want to exploit the structural features of these highly regulated states in order to design novel antibiotics that inhibit gene transcription for drug therapies as this system, although important for responding to stress, is not essential for normal bacterial growth under a range of conditions, but is important for many adaptations in hostile environments such as the host. The bacteria therefore will be under less pressure to develop resistance. This approach is especially effective when combined with other antibiotics. Inhibiting bacterial RNAP, and hence gene transcription, is a validated antibiotic strategy e.g. in controlling TB infections, so our work should provide novel avenues for effective antibiotic development at a time when it is crucial to have new reagents to control dangerous pathogenic bacteria of humans and animals.

Technical Summary

RNA polymerase (RNAP) is a fundamental cellular machinery responsible for gene transcription. RNAP is conserved from bacteria to humans. Gene transcription is a highly regulated event in response to cues in development, growth and many varying environmental stimuli. Although significant advance has been made towards understanding how RNAP functions as an enzyme, how RNAP is controlled by in cis and in trans acting factors, and more importantly, how these control factors and the RNA polymerase are co-organised on the DNA, is still poorly understood. Establishing the molecular organisation of complete gene control systems is critical to our understanding of the RNAPs' sensing the outputs of signal transduction pathways and for studying gene regulatory systems in the context of chromosomes and in the intact cell.

In our work we employ the bacterial RNAP and its major variant sigma factor sigma54 as a simplified tractable model system, important in many bacteria, to study a strategy by which RNAP stays in an inhibited state. Specific activator proteins acting remotely from RNAP at enhancer like DNA sequences far removed from transcription start site are the AAA+ ATPases that then convert the RNAP from an inactive state to a transcriptionally competent enzyme to achieve regulated gene expression. Here, we plan to utilise the latest development in cryo electron microscopy and our acquired biochemical knowledge and reagents in a number of exemplar gene regulation systems to study how these systems are organised on the DNA and how they are regulated by upstream control signals. This is important in our understanding of many protein-DNA complexes and leads us a step closer to study these systems in a cellular context. Bacterial RNAP is a validated antimicrobial target, and some of the controlling hotspots we identified in RNAP are not targeted by current antibiotics. So our work should provide novel avenues for new effective antibiotic developments against pathogens.

Planned Impact

Key groups who will be impacted upon by the proposed research are:

(i) Academics: The academic sector will be the main short to medium term beneficiary, as the proposed research will provide knowledge, reagents and new structures of several ATPase driven gene regulation response systems in E. coli, a major studied bacterium widely used to unravel the basic life processes for many decades. Furthermore, the project will provide a clear opportunity for career development and training of individuals, both nationally and internationally.Importantly in vitro mechanistic data from new structures and biochemistry will be used to help tackle controlling gene expression in vivo for antimicrobials developments .
(ii) Society at large: Benefits to society at large will be twofold: In the short term, the proposed project will provide employment and training for individuals at the postdoctoral level providing experience of project design, management as well as its high level scientific implementation, thereby directly contributing to the national economy. The interdisciplinary nature of the proposed research will greatly enhance training of the associated PDRAs, especially with respect to their ability to work within large interdisciplinary teams and deploy cutting edge approaches. Longer term benefits include impacts on health care through stimulating the formulation of new antimicrobials and refining the usage of existing ones.
(iii) Industry: The industrial sector is another potential medium to long-term beneficiary. The proposed research will generate knowledge that could potentially be exploited for new product development by the biotech and agri tech industries (e.g. against therapeutically proven antimicrobial targets, and through use of synthetic biology approaches). Research results could potentially identify novel targets for therapeutic intervention at protein/RNA, protein/protein and protein/DNA interaction level. The IC Business Development teams 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 teams in the area of translating research into marketable products.
(iv) Government: One of the remits of the new IC Institute for Global Health is to translate new scientific knowledge into applications to improve global health by influencing international policy. Expertise offered by the IC Institute for Global Health could therefore be exploited for using discoveries made as a result of the proposed research to inform future health care policies.

Exploitation and Application: A number of structures exist within ICL for exploitation of knowledge gained and the development of beneficial applications. For example, we will make use of the expertise offered by ICL Innovations teams 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. Results from the project will provide opportunities for novel drug-target discovery centered around protein/RNA, protein/protein and protein/DNA interactions and links to the synthetic biologists.

For drug discovery, and noting the diminishing content of the pipelines that feed this aim, one possibility is that, as a result of the proliferation of technologies intended to enable drug discovery, the basic biological questions are being overlooked or ignored. Technological development in high throughput target identification, screening, library synthesis, and validation have their place, but they are essentially just tools, and a clear understanding of the underlying biology is paramount.This project affords such a deep mechanistic understanding of cellular responses that can then frame new approaches to drug discovery.
Description There have been a series of setbacks since the grant started in Oct 2018. However, we have made the following progress:

To understand the exact roles of activators and the mechanisms of DNA opening, we have overcome technical challenges and have now obtained a high resolution structure (4 Å) of the activator protein ( PspF AAA+ domain) in complex with RNAP-sigma54-DNA with regions in PspF and RNAP-sigma54 reaching 2.8- 3 Å. As such, we could distinguish differing nucleotide states of individual subunits as well as how each subunit interacts with sigma54 and DNA, providing molecular details of a key functional intermediate state. Importantly we observe how DNA is opened up precisely and what are the roles of AAA+ activators in DNA opening. Our results reveal novel interaction modes between DNA and protein and a unique mechanism of DNA opening. We are in the process of submitting these results to a leading international journal for publication.

To understand how the activator bounds complex is organised at the upstream enhancer DNA site, we have successfully obtain the complete complex between the RNA repair protein RtcR, the promoter-enhance DNA, RNA polymerase, sigma54, Integrated Host Factor, thus assembling the complete transcription activation complex which hitherto has yet to be revealed. We have obtained a medium resolution structure which reveals how three pairs of RtcR bind at the DNA while forming a hexameter. This will provide a first structural model on how these proteins bind adjacent to linear DNA while forming hexameters. Further, we have developed new tools and protocols to overcome preferential orientation issues on cryoEM grids, increase the instability of the complex and use a new cryoEM grid types. We hope to obtain high resolution structure of the complete complex in the near future, which will will provide a comprehensive understanding of how bacterial enhancer-binding protein recognises and organises at Upstream Activation Sequence and how it engages with RNAP-sigma54-DNA.
Exploitation Route The outcome will provide fundamental mechanistic insights into DNA opening, transcription initiation and protein-DNA interactions at the promoter-enhancer sites. Some of these will challenge established dogma and open up new research directions and while some of the knowledge (such as the organisation of enhancer-bound transcription complex, DNA opening/melting mechanisms) will likely become text-book information.
Sectors Education,Environment,Healthcare

Description We provide cryoEM maps and structural models of bacteriophage protein OCR in complex with RNAP 
Type Of Material Database/Collection of data 
Year Produced 2020 
Provided To Others? Yes  
Impact Our work provides a molecular basis for how bacteriophage protein OCR inhibits transcription of bacteria, thus allowing bacteriophage to successfully infect bacteria. The structures suggest possible strategies that could be exploited in adopting DNA mimicry and forms a basis for novel antibiotics development. 
Title RamA-RNAP transcription complexes 
Description cryoEM maps and models of RamA-dependent transcriptional complexes at class I and class II promoter sites. 
Type Of Material Database/Collection of data 
Year Produced 2021 
Provided To Others? Yes  
Impact It unravels how RamA recruits RNAP machinery to the two genes involved in antibiotic resistance, thus providing structural and molecular basis for RamA-dependent antibiotic resistance. 
Title cryoEM maps and models of INO80 
Description We provide cryoEM maps and structural models of the human INO80 chromatin remodeller, revealing first structures of a multi-subunit chromatin remodeller. 
Type Of Material Database/Collection of data 
Year Produced 2018 
Provided To Others? Yes  
Impact These structures reveal the architecture of the INO80 complex, including Ino80 and actin-related proteins, which is assembled around a single RUVBL1 (Tip49a) and RUVBL2 (Tip49b) AAA+ heterohexamer. An unusual spoked-wheel structural domain of the Ino80 subunit is engulfed by this heterohexamer; both, in combination, form the core of the complex. We also identify a cleft in RUVBL1 and RUVBL2, which forms a major interaction site for partner proteins and probably communicates these interactions to its nucleotide-binding sites. We thus reveal the roles of the AAA+ protein RUVBL1 and RUVBL2. 
Title cryoEM maps and models of RNAP transcription initiation intermediate complexes 
Description We provide cryoEM maps and structural models of an intermediate complex caught during transcription initiation as well as open promoter complex and a initial transcribing complex. 
Type Of Material Database/Collection of data 
Year Produced 2018 
Provided To Others? Yes  
Impact Gene transcription is carried out by multi-subunit RNA polymerases (RNAPs). Transcription initiation is a dynamic multi-step process that involves the opening of the double-stranded DNA to form a transcription bubble and delivery of the template strand deep into the RNAP for RNA synthesis. We capture a new intermediate state at 4.1 Å where promoter DNA is caught at the entrance of the RNAP cleft. Combining with new structures of the open promoter complex and an initial de novo transcribing complex at 3.4 and 3.7 Å, respectively, our studies reveal the dynamics of DNA loading and mechanism of transcription bubble stabilization that involves coordinated, large-scale conformational changes of the universally conserved features within RNAP and DNA. In addition, our studies reveal a novel mechanism of DNA strand separation. 
Title cryoEM maps and structures of RNA polymerase closed and intermediate complexes 
Description We provide cryoEM maps and structures of bacterial transcription complexes containing the major variant sigma factors, in particular the closed and intermediate complex. 
Type Of Material Database/Collection of data 
Year Produced 2017 
Provided To Others? Yes  
Impact Gene transcription is carried out by RNA polymerases (RNAPs). For transcription to occur, the closed promoter complex (RPc), where DNA is double stranded, must isomerize into an open promoter complex (RPo), where the DNA is melted out into a transcription bubble and the single-stranded template DNA is delivered to the RNAP active site. Using a bacterial RNAP containing the alternative s54 factor and cryoelectron microscopy, we determined structures of RPc and the activator-bound intermediate complex en route to RPo at 3.8 and 5.8 Å. Our structures show how RNAP-s54 interacts with promoter DNA to initiate the DNA distortions required for transcription bubble formation, and how the activator interacts with RPc, leading to significant conformational changes in RNAP and s54 that promote RPo formation. We propose that DNA melting is an active process initiated in RPc and that the RNAP conformations of intermediates are significantly different from that of RPc and RPo. 
Description Durham 
Organisation Durham University
Country United Kingdom 
Sector Academic/University 
PI Contribution Carrying out scientific research and reporting the results via publication
Collaborator Contribution providing reagents and knowledge
Impact Publication: Ye et al. eLife 2020.
Start Year 2018
Description New collaboration with University of Sheffield 
Organisation University of Sheffield
Country United Kingdom 
Sector Academic/University 
PI Contribution We have provided reagents to the partner and helped the partner to complete a research and this has yielded a publication (in press, Nucleic Acids Research)
Collaborator Contribution They have conducted the majority of the research
Impact Joint publication
Start Year 2021
Description School lecture 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact I gave a lecture to A level students at a girl's grammar school. I talked about my journey into science and my current career and blended in with my research and science. Questions and answers session followed this and it was very positive and indeed a number of students emailed me immediately afterwards asking about work experience in my lab. Unfortunately this event had to be conducted online due to COVID19.
Year(s) Of Engagement Activity 2021
Description Talk to private donor 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact I was approached by a potential private donor - due to our work advertised online and through funding bodies. I spoke to them about what we do and what the potential impact might be. Subsequently I received £12500 private donation to my research. The donors even suggested to have a drive to get more donations to my work. I was very touched and proud that our research has inspired general public in such a way.
Year(s) Of Engagement Activity 2021
Description hosting school students 
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 Annually I host 2-4 school and undergraduate students for work experience in my lab to encourage them to pursue science at degree and postgraduate levels
Year(s) Of Engagement Activity 2017,2018,2019,2020