Organisation and regulation of bacterial enhancer-binding proteins

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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.