Structure of the bacteriophage PhiKZ non-virion RNA polymerase.

Bacteriophage ΦKZ (PhiKZ) is the archetype of a family of massive bacterial viruses. It is considered to have therapeutic potential as its host, Pseudomonas aeruginosa, is an opportunistic, intrinsically antibiotic resistant, pathogen that kills tens of thousands worldwide each year. ΦKZ is an incredibly interesting virus, expressing many systems that the host already possesses. On infection, it forms a 'nucleus', erecting a barrier around its genome to exclude host endonucleases and CRISPR-Cas systems. ΦKZ infection is independent of the host transcriptional apparatus. It expresses two different multi-subunit RNA polymerases (RNAPs): the virion RNAP (vRNAP) is injected with the viral DNA during infection to transcribe early genes, including those encoding the non-virion RNAP (nvRNAP), which transcribes all further genes. ΦKZ nvRNAP is formed by four polypeptides thought to represent homologues of the eubacterial ß/ß' subunits, and a fifth with unclear homology, but essential for transcription. We have resolved the structure of ΦKZ nvRNAP to better than 3.0 Å, shedding light on its assembly, homology, and the biological role of the fifth subunit: it is an embedded, integral member of the complex, the position, structural homology and biochemical role of which imply that it has evolved from an ancestral homologue to σ-factor.

The study of the phiKZ phage non-canonical non-virion RNA polymerase.

Non-canonical multisubunit DNA-dependent RNA-polymerases (RNAP) form a new group of the main transcription enzymes, which have only distinct homology to the catalytic subunits of canonical RNAPs of bacteria, archaea and eukaryotes. One of the rare non-canonical RNAP, which was partially biochemically characterized, is non-virion RNAP (nvRNAP) encoded by Pseudomonas phage phiKZ. PhiKZ nvRNAP consists of five subunits, four of which are homologs of ß and ß' subunit of bacterial RNAP, and the fifth subunits with unknown function. To understand the role of the fifth subunit in phiKZ nvRNAP, we created co-expression system allowing to get recombinant full five-subunit (5s) and four-subunit (4s) complexes and performed their comparison. The 5s recombinant complex is active on phage promoters in vitro as the native nvRNAP. The 4s complex cannot extend RNA, so 4s complex is not a catalytically active core of phiKZ nvRNAP. Thus, the phiKZ fifth subunit is not only a promoter-recognition subunit, but it plays an important role in the formation of active phiKZ nvRNAP.