Characterization of the rpoC gene of Streptomyces coelicolor A3(2) and its use to develop a simple and rapid method for the purification of RNA polymerase.
The Streptomyces coelicolor rpoC gene, that encodes the beta' subunit of RNA polymerase, was isolated using the Escherichia coli rpoC gene as a hybridization probe. Comparison of the predicted amino acid sequence of the S. coelicolor beta' subunit to those characterized from other bacteria revealed three distinct subfamilies of beta' subunits, one of which consists of the S. coelicolor subunit and those from Mycobacterium leprae and Mycoplasma genitalium. Using site-directed mutagenesis, the carboxy terminus of the S. coelicolor beta' subunit was modified to contain six histidine residues. The histidine-tagged gene, rpoCHIS, was used to replace the wild-type allele in the chromosome of S. coelicolor and S. lividans. These strains were unaffected in growth and sporulation, demonstrating that the histidine-tagged RNA polymerase was competent to carry out all essential in-vivo functions. During a 1-day procedure, highly purified RNA polymerase was obtained by nickel-NTA agarose affinity chromatography followed by heparin-sepharose chromatography. Using in-vitro run-off transcription assays, the affinity purified RNA polymerase was shown to initiate transcription correctly from the S. lividans galP1 and galP2 promoters, and the Bacillus subtilus veg and ctc promoters. An extension of this procedure yielded highly-purified core RNA polymerase. To facilitate introduction of the rpoCHIS allele into other genetic backgrounds, a mutation in the adjacent gene, rpoB (rifA), conferring rifampin-resistance, was isolated in S. coelicolor to provide a genetic marker to follow transfer of the rpoCHIS allele. The use of this affinity chromatography procedure, in combination with the ability to introduce the rpoCHIS allele into different Streptomyces strains by transformation, will greatly facilitate the in-vitro analysis of transcription in members of this genus.
Babcock
M J
MJ
Department of Gene Expression Sciences, SmithKline Beecham, King of Prussia, PA 19406, USA.
Buttner
M J
MJ
Keler
C H
CH
Clarke
B R
BR
Morris
R A
RA
Lewis
C G
CG
Brawner
M E
ME
D000595:Amino Acid Sequence; D001483:Base Sequence; D002846:Chromatography, Affinity; D002876:Chromosomes, Bacterial; D003001:Cloning, Molecular; D012321:DNA-Directed RNA Polymerases / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D006639:Histidine; D008969:Molecular Sequence Data; D009154:Mutation; D009532:Nickel; D010940:Plant Proteins / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011995:Recombination, Genetic; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013302:Streptomyces / Q000737:chemistry / Q000235:genetics*
D010940:Plant Proteins; D011994:Recombinant Proteins; C101305:RpoB protein, Oryza sativa; D006639:Histidine; D009532:Nickel; C108570:beta' subunit of RNA polymerase; D012321:DNA-Directed RNA Polymerases