In vitro transcription system using reconstituted RNA polymerase (Eσ 70 , Eσ H , Eσ E and Eσ S ) of Pseudomonas aeruginosa

Abstract

We have developed an in vitro transcription system for Pseudomonas aeruginosa genes, using RNA polymerase (RNAP) holoenzyme reconstituted with purified sigma protein and RNAP core enzyme. The RNAP core enzyme was directly purified from P. aeruginosa PAO1 cells. The sigma factors of P. aeruginosa (sigma(70), sigma(H), sigma(E) and sigma(S)) were prepared in a hexa-histidine tagged form, which were expressed in Escherichia coli and purified using a HisTrap Chelating column. The RNAP holoenzyme reconstituted from core enzyme with each sigma factor recognized correctly each of the cognate promoters. This system will be useful for the promoter analysis of many genes in P. aeruginosa.