Template strand deoxyuridine promoter recognition by a viral RNA polymerase

Abstract

Bacillus subtilis bacteriophage AR9 employs two strategies for efficient host takeover control and host antiviral defense evasion – it encodes two unique DNA-dependent RNA polymerases (RNAPs) that function at different stages of virus morphogenesis in the cell, and its double stranded (ds) DNA genome contains uracils instead of thymines throughout1,2. Unlike any known RNAP, the AR9 non-virion RNAP (nvRNAP), which transcribes late phage genes, recognizes promoters in the template strand of dsDNA and efficiently differentiates obligatory uracils from thymines in its promoters3. Here, using structural data obtained by cryo-electron microscopy and X-ray crystallography on the AR9 nvRNAP core, holoenzyme, and a promoter complex, and a variety of in vitro transcription assays, we elucidate a unique mode of uracil-specific, template strand-dependent promoter recognition. It is achieved by a tripartite interaction between the promoter specificity subunit, the core enzyme, and DNA adopting a unique conformation. We also show that interaction with the non-template strand plays a critical role in the process of AR9 nvRNAP promoter recognition in dsDNA, and that the AR9 nvRNAP core and a part of its promoter specificity subunit that interacts with the core are structurally similar to their bacterial RNAP counterparts. Our work demonstrates the extent to which viruses can evolve new functional mechanisms to control acquired multisubunit cellular enzymes and make these enzymes serve their needs.