Organisation and regulation of bacterial enhancer-binding proteins

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.

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.

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.