An improved procedure for the purification of the Escherichia coli RNA polymerase omega subunit.
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.
Vrentas
Catherine E
CE
Department of Bacteriology, University of Wisconsin-Madison, Madison, WI 53706, USA.
Gaal
Tamas
T
Burgess
Richard R
RR
Gourse
Richard L
RL
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*
C111232:RNA polymerase omega subunit; D012321:DNA-Directed RNA Polymerases