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
