Structure-based functional analysis of RNA Polymerase

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Research project summaryGene expression, the basic requirement for life, is considered the central dogma of molecular biology. This dogma describes the directional flow of genetic information from deoxyribonucleic acid (DNA) to ribonucleic acid (RNA) (transcription), to polypeptide chains (translation) that undergo post-translational modification to form functional proteins. Bacterial RNA polymerase (RNAp) is the central enzyme of gene expression whereby it accomplishes the polymerization of ribonucleoside triphosphates (NTPs). The catalytic core enzyme consists of five subunits In bacteria, RNAp, interacts with other cellular machineries, such as translation, DNA repair and replication. Bacterial RNAp is also a potent target for the development of antibacterials. RNAps in all living organisms are multisubunit, multidomain enzymes, and the functions of all the domains are still not well understood. Our laboratory solved structures of multisubunit RNAp from Escherichia coli (E.coli) stalled at a DNA lesion, which highlighted movements of some particular domains, which may be involved in signalling to translation and DNA repair machineries. Our laboratory also solved the structure of a small single-subunit relative of multi-subunit RNAps, which highlighted the structural conservation of domains of the active center, but divergence in the rest of the molecule, thus prompting the investigation of structure-based functional comparisons.Aims and objectivesThe overall aim of the project is to develop and characterize new inhibitors of RNAp. The project proposes to perform structure-based functional analysis of multisubunit RNAp and its single-subunit relative to bring insight into: the cross-talk between RNAp, ribosome, and DNA repair factors; functional conservation of the RNAp structure, principles of transcription inhibition by other cellular machineries and small molecules.The project will apply a wide arsenal of biochemical, microbiological, genetics, and structural biology techniques. We will use a range of known and new inhibitors of transcription as molecular probes of the functions of RNAps. Besides fundamental insights into the functions of bacterial RNAp, the project may lead to the discovery of new targets for inhibitors and novel candidate molecules for future development as antibacterials.KEYWORDSCell biology: understanding the fundamental properties, structure and function of the cell and how it responds to and influences its local environmentStructural biology and biophysics: understanding the atomic organisation of molecules and macromolecular complexes and the dynamic, functional relationships between these components in cells and biological systemsAntimicrobial resistance