Rapid RNA polymerase genetics: one-day, no-column preparation of reconstituted recombinant Escherichia coli RNA polymerase.
We present a simple, rapid procedure for reconstitution of Escherichia coli RNA polymerase holoenzyme (RNAP) from individual recombinant alpha, beta, beta', and sigma 70 subunits. Hexahistidine-tagged recombinant alpha subunit purified by batch-mode metal-ion-affinity chromatography is incubated with crude recombinant beta, beta', and sigma 70 subunits from inclusion bodies, and the resulting reconstituted recombinant RNAP is purified by batch-mode metal-ion-affinity chromatography. RNAP prepared by this procedure is indistinguishable from RNAP prepared by conventional methods with respect to subunit stoichiometry, alpha-DNA interaction, catabolite gene activator protein (CAP)-independent transcription, and CAP-dependent transcription. Experiments with alpha (1-235), an alpha subunit C-terminal deletion mutant, establish that the procedure is suitable for biochemical screening of subunit lethal mutants.
Tang
H
H
Department of Chemistry, Rutgers University, New Brunswick, NJ 08855, USA.
Severinov
K
K
Goldfarb
A
A
Ebright
R H
RH
D001483:Base Sequence; D002846:Chromatography, Affinity; D004247:DNA / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D016324:Sequence Tagged Sites; D013997:Time Factors; D014158:Transcription, Genetic
D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D004247:DNA; D012321:DNA-Directed RNA Polymerases