Abstract
Infection of Escherichia coli by the T7 phage leads to rapid and selective inhibition of the bacterial RNA polymerase (RNAP) by the 7 kDa T7 protein Gp2. We describe the identification and functional and structural characterisation of a novel 7 kDa T7 protein, Gp5.7, which adopts a winged helix-turn-helix-like structure and specifically represses transcription initiation from host RNAP-dependent promoters on the phage genome via a mechanism that involves interaction with DNA and the bacterial RNAP. Whereas Gp2 is indispensable for T7 growth in E. coli, we show that Gp5.7 is required for optimal infection outcome. Our findings provide novel insights into how phages fine-tune the activity of the host transcription machinery to ensure both successful and efficient phage progeny development.
Highlights
Many bacteriophages use diverse mechanisms to repurpose, redirect or inhibit the bacterial transcription machinery, the RNA polymerase (RNAP), to coordinate phage gene expression and developmental needs during infection [1,2]
The principal finding of this study is that T7 requires, in addition to Gp2, another RNAP inhibiting small protein to temporally control the activity of the host RNAP during infection
Infection of Escherichia coli (Ec) by T7 begins with the translocation of the left end of the T7 genome into the host which is coupled to the transcription from T7 early genes from T7 A1-A3 promoters by the Ec RNAP
Summary
Many bacteriophages (phages) use diverse mechanisms to repurpose, redirect or inhibit the bacterial transcription machinery, the RNA polymerase (RNAP), to coordinate phage gene expression and developmental needs during infection [1,2]. And middle genes generally encode proteins required for phage RNA synthesis, DNA replication and host takeover; the late genes specify T7 virion assembly and structural proteins. The host RNAP is shut off by the coordinated action of the early gene product Gp0.7 and the essential middle gene product Gp2; the viral single-subunit RNAP (T7 RNAP, Gp1, a product of an early gene) transcribes the middle and late viral genes. The shutting down of host RNAP is crucial for the successful completion of the infection cycle: Gp0.7 is a protein kinase that phosphorylates the Ec RNAP, leading to increased termination of transcription at sites located between the early and middle genes on the T7 genome [6]; and Gp2 binds in the main DNA binding channel in Ec RNAP and thereby prevents the formation of the transcriptionally proficient open promoter complex (RPo) at the T7 A1-3 promoters [7]. Gp2 is indispensable for T7 growth and its absence leads to interference by the host RNAP transcription initiated from T7 A1-3 promoters, with middle and late T7 gene transcription by the T7 RNAP [8]
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