Open AccessIn vitro transcription system using reconstituted RNA polymerase (Eσ 70 , Eσ H , Eσ E and Eσ S ) of Pseudomonas aeruginosa
Author(s) -
Fujita Masaya,
Sagara Yasuhiro,
Aramaki Hironori
Publication year - 2000
Publication title -
fems microbiology letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.899
H-Index - 151
eISSN - 1574-6968
pISSN - 0378-1097
DOI - 10.1111/j.1574-6968.2000.tb08967.x
Subject(s) - rna polymerase , sigma factor , microbiology and biotechnology , transcription (linguistics) , pseudomonas aeruginosa , promoter , biology , polymerase , gene , escherichia coli , enzyme , chemistry , biochemistry , genetics , gene expression , bacteria , linguistics , philosophy
We have developed an in vitro transcription system for Pseudomonas aeruginosa genes, using RNA polymerase (RNAP) holoenzyme reconstituted with purified σ protein and RNAP core enzyme. The RNAP core enzyme was directly purified from P. aeruginosa PAO1 cells. The σ factors of P. aeruginosa (σ 70 , σ H , σ E and σ 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 σ factor recognized correctly each of the cognate promoters. This system will be useful for the promoter analysis of many genes in P. aeruginosa .