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Title: Characterisation of bacteriophage-encoded inhibitors of the bacterial RNA polymerase
Author: Sheppard, Carol Maria
ISNI:       0000 0004 2735 4861
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2013
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RNA polymerase (RNAP) is an essential enzyme which catalyses transcription; a highly regulated process. Bacteriophage are viruses which infect bacteria and as a result have evolved a diverse range of mechanisms to regulate the bacterial RNAP to serve the needs of the virus. T7 Gp2 and Xp10 P7 are two bacteriophage-encoded transcription factors that inhibit the activity of the bacterial RNAP. The aim of this study is to investigate the molecular mechanisms of action of Gp2 and P7. Fluorescence anisotropy experiments proved Gp2 to bind to RNAP, independently of the σ- factor, with a 1:1 stoichiometry and a low nanomolar affinity. In vitro transcription assays demonstrated that a negatively charged strip in Gp2 is the major determinant for its inhibitory activity. Furthermore, it was shown that efficient Gp2-mediated inhibition of RNAP also depends upon the highly negatively charged and flexible σ70 specific domain, R1.1. Gp2 and R1.1 both bind in the downstream-DNA binding channel and exert long-range antagonistic effects on RNAP-promoter DNA interactions around the transcription start site. A systematic mutagenesis screen was used to identify residues in P7 necessary for binding to the RNAP; results were interpreted in the context of a newly resolved NMR structure of P7. Electrophoretic mobility shift assays revealed that P7 ‘traps’ a RNAP-promoter DNA complex en route to the transcriptionally-competent complex. Preliminary results from a fluorescence based RNAP-DNA interaction assay suggest that P7 may target RNAP interactions with the -35 promoter element and the ‘discriminator region’. This study has contributed to our understanding of how non-bacterial transcriptional factors can influence bacterial gene expression by modulating RNAP activity. This study has also uncovered vulnerabilities in RNAP, which have the potential to be exploited therapeutically. To this end, these structure-function studies of Gp2 and P7 have provided the basis for the rational design of novel anti-bacterial compounds.
Supervisor: Wigneshweraraj, Sivaramesh Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral