title: An improved procedure for the purification of the Escherichia coli RNA polymerase omega subunit.
abstract: We report an improved procedure for purification of the omega subunit of Escherichia coli RNA polymerase. In contrast to the original procedure, the revised procedure: (i) allows purification of omega entirely from the soluble fraction, obviating the need for denaturation/renaturation, (ii) results in >99% pure omega in only two chromatographic steps, and (iii) improves the yield of purified omega by at least 5-fold. Reconstitution of E. coli RNAP from omega purified by this procedure, as well as purified sigma and core RNAP lacking omega, produces active holoenzyme in vitro, and co-overexpression of omega from a plasmid containing rpoZ and an additional plasmid encoding the other RNAP core subunits results in production of active core enzyme in vivo.
authors: Department of Bacteriology, University of Wisconsin-Madison, Madison, WI 53706, USA.
authors: Catherine E
authors: CE
authors: Vrentas
authors: Tamas
authors: T
authors: Gaal
authors: Richard R
authors: RR
authors: Burgess
authors: Richard L
authors: RL
authors: Gourse
mesh_terms: D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification; D010957:Plasmids / Q000235:genetics* / Q000378:metabolism*
chemical_list: C111232:RNA polymerase omega subunit; D012321:DNA-Directed RNA Polymerases