Structure and activity of the giant RNA polymerase ejected from Pseudomonas phage DEV

A hallmark of Schitoviridae bacteriophages is a giant (∼3,500-residue) virion-associated RNA polymerase (vRNAP) that is ejected into the host to initiate early transcription. Structural and functional characterization of vRNAP has thus far been limited to coliphage N4, leaving the conservation of vRNAP architecture, promoter recognition, and regulation across Schitoviridae largely unexplored. Here, we characterized the vRNAP from the schitovirus DEV, a lytic phage of Pseudomonas aeruginosa used in experimental phage therapy cocktails. Cryo-EM structures of DEV vRNAP (gp71) in the apo state and bound to its cognate ssDNA hairpin promoter reveal that DEV vRNAP adopts a conserved closed-fist, single-subunit RNA polymerase fold and recognizes a partially folded ssDNA hairpin promoter, similar to phage N4 vRNAP, despite sharing only 20% sequence identity. The ∼140-kDa C-terminal domain (CTD) adopts a trapezoidal, predominantly α-helical architecture comprising two subdomains with only weak structural similarity to the N4 CTD (RMSD ∼15.3 Å). The RNAP domain and CTD are connected by a short linker but remain highly dynamic, forming a compact assembly in only a small fraction of particles, whereas the N-terminal domain, which has proposed membrane-binding activity, was not resolved. Radiolabeled runoff transcription assays demonstrated that the CTD strongly inhibits transcription both intramolecularly, in cis, and as an isolated domain, in trans. Together, these findings reveal that DEV and N4 vRNAPs conserve a common RNAP fold, promoter-recognition mechanism, and CTD-mediated autoinhibition despite substantial sequence and structural divergence, defining conserved principles of Schitoviridae vRNAP architecture and function.