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 beta and beta' 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. (C) 2019 Elsevier Inc. All rights reserved.