Coexpression of omega 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 alpha 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 omega, 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 omega had a 67% increase compared with that of one lacking omega, indicating that omega promotes the assembly of Xcc core RNAP. In addition, Xcc core RNAP lacking omega showed a 13-fold decrease in enzymatic activity in comparison with that containing omega. Promoter-specific transcription assays by recombinant Xcc core RNAP reconstituted with external added sigma factor showed that the absence of omega debilitates the transcriptional activity of Xcc RNAP. Our results demonstrated that omega is not only capable of strengthening the stability, but is also required for the maintenance of enzymatic activity of Xcc RNAP.
Cheng
Ching-Yuan
CY
Institute of Molecular Biology, National Chung Hsing University, Taichung 40227, Taiwan, Republic of China.
Yu
Yu-Jen
YJ
Yang
Ming-Te
MT
D003001:Cloning, Molecular; D012321:DNA-Directed RNA Polymerases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism*; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D011994:Recombinant Proteins / Q000302:isolation & purification; D014158:Transcription, Genetic; D016959:Xanthomonas campestris / Q000201:enzymology*
D011994:Recombinant Proteins; C111232:RNA polymerase omega subunit; D012321:DNA-Directed RNA Polymerases