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

We have developed an in vitro transcription system for Pseudomonas aerugino sa genes, using RNA polymerase (RNAP) holoenzyme reconstituted with purifie d sigma protein and RNAP core enzyme. The RNAP core enzyme was directly pur ified from P. aeruginosa PAO1 cells. The sigma factors of P. aeruginosa (si gma(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 enzy me with each sigma factor recognized correctly each of the cognate promoter s. This system will be useful for the promoter analysis of many genes in P. aeruginosa. (C) 2000 Federation of European Microbiological Societies. Pub lished by Elsevier Science B.V. All rights reserved.