Structures and Mechanisms of RNA polymerase inhibition and activation

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RNA polymerase 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 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. 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 RNAP itself is controlled by factors that signal special cellular states and events, is still poorly understood. We use the bacterial RNAP and its major variant sigma factor, sigma54, as a simplified model system to study how RNAP stays in an inhibited state and how activator proteins, acting remotely from where transcription will start, utilise cellular energy in the form of a high energy molecule called ATP, to convert the RNAP from an inactive enzyme to a transcriptionally competent enzyme. We have just determined the crystal structure of RNAP-sigma54 so that we now have a detailed view of what RNAP-sigma54 looks like. Our structure explains how sigma54 maintains RNAP in an inhibited state. Furthermore, we discovered many of the inhibitory strategies we see are shared to some extent by other bacterial and eukaryotic factors and reveal there are conserved hotspots in RNAP that are targeted to varying degrees by different elements and transcriptional factors to fine-tune transcription inhibition. In this current proposed research, we plan to utilise our newly acquired knowledge, experience and reagents already generated to address fundamental questions on how this inhibited state is relieved by activator. This is likely to shed light into how RNAP in humans, plants and animals is activated. Furthermore, we want to exploit the structural features of the inhibited state to design novel antibiotics that inhibit gene transcription by attacking important sites and surfaces on the bacterial RNAP enzyme that have not been targeted before for drug therapies. Inhibiting bacterial RNAP, and hence gene transcription, is a validate 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.