Coexpression of ω subunit in E. coli is required for the maintenance of enzymatic activity of Xanthomonas campestris pv. campestris RNA polymerase

In this study, plasmid pBBad22K was modulated to be able to coexpress the subunits of Xanthomonascampestris pv. campestris (Xcc) core RNA polymerase (RNAP) in an Escherichia coli host. The subunit-encoding genes of Xcc core RNAP were PCR-amplified respectively to convert into gene cassettes in which an intact subunit-encoding gene and a ribosome binding site (RBS) preceding the gene were contained, and were then cloned one by one into pBBad22K. In addition, a hexahistidine tag (His-tag) was introduced into the C-terminus of α subunit-encoded rpoA during PCR for facilitating purification of Xcc core RNAP. The resultant vectors, pBC-CBA and pBC-CBAZ, were used for overproduction of Xcc core RNAP lacking or containing ω, respectively. The assembly of Xcc core RNAP subunits that were coexpressed from these vectors was demonstrated after purification via two-step column chromatography. The yield of Xcc core RNAP containing ω had a 67% increase compared with that of one lacking ω, indicating that ω promotes the assembly of Xcc core RNAP. In addition, Xcc core RNAP lacking ω showed a 13-fold decrease in enzymatic activity in comparison with that containing ω. Promoter-specific transcription assays by recombinant Xcc core RNAP reconstituted with external added σ factor showed that the absence of ω debilitates the transcriptional activity of Xcc RNAP. Our results demonstrated that ω is not only capable of strengthening the stability, but is also required for the maintenance of enzymatic activity of Xcc RNAP.