Abstract
Bacteriophage T7 infects Escherichia coli and evades the host restriction/modification system. The Ocr protein of T7 was shown to exist as a dimer mimicking DNA and to bind to host restriction enzymes, thus preventing the degradation of the viral genome by the host. Here we report that Ocr can also inhibit host transcription by directly binding to bacterial RNA polymerase (RNAP) and competing with the recruitment of RNAP by sigma factors. Using cryo electron microscopy, we determined the structures of Ocr bound to RNAP. The structures show that an Ocr dimer binds to RNAP in the cleft, where key regions of sigma bind and where DNA resides during transcription synthesis, thus providing a structural basis for the transcription inhibition. Our results reveal the versatility of Ocr in interfering with host systems and suggest possible strategies that could be exploited in adopting DNA mimicry as a basis for forming novel antibiotics.
Highlights
Bacteriophage T7 infects Escherichia coli and hijacks the host cellular machinery to replicate its genome (Studier, 1972; Kruger and Schroeder, 1981; Hausmann and Messerschmid, 1988)
An early study by Ratner (1974) showed that upon T7 infection, Overcome Classical Restriction (Ocr) protein was detected in pulldown experiments using host RNA polymerase (RNAP) as bait, suggesting that Ocr could associate with the host transcriptional machinery
Our observed affinity of Ocr for RNAP is about three orders of magnitude lower than the values reported for the association of Ocr with the EcoKI type I restriction modification enzyme (Atanasiu et al, 2002) as would be expected if the type I RM enzyme was the primary target for Ocr
Summary
Bacteriophage T7 infects Escherichia coli and hijacks the host cellular machinery to replicate its genome (Studier, 1972; Kruger and Schroeder, 1981; Hausmann and Messerschmid, 1988). Proteins gp0.7, gp and gp5.7 inhibit cellular transcription (Camara et al, 2010; TabibSalazar et al, 2018) whereas gp0.3 inhibits restriction/modification (RM) enzymes (Studier, 1975). Ocr is abundantly expressed and forms a dimer that mimics the structure of a slightly bent 20 base pair B-form DNA (Issinger and Hausmann, 1972; Walkinshaw et al, 2002) and blocks the DNA binding grooves of the type I RM enzyme, preventing the degradation and modification of the T7 genome by the host. Since Ocr is a DNA mimicry protein, it is possible that the abundantly expressed Ocr (estimated to be several hundreds of molecules per cell at least) (Hausmann and Messerschmid, 1988) interferes with other DNA processing systems of the host. Early evidence of an interaction between Ocr and the host RNA polymerase (RNAP) was obtained using pull-down affinity chromatography (Ratner, 1974)
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