Rapid isolation of RNA polymerase from sporulating cells of Bacillus subtilis.
A highly ordered program of temporal and spatial gene activation during sporulation in Bacillus subtilis is governed by the principal RNA polymerase, and RNA polymerases containing at least five developmental sigma factors appearing successively during sporulation. This report describes a rapid procedure for extracting RNA polymerase from sporulating B. subtilis cells, which involves the construction of hexahistidine tagged beta' subunit of RNA polymerase and the isolation of RNA polymerase holoenzyme with Ni2+-NTA resin. In in vitro transcription of various promoters with the RNA polymerase thus purified, we observed the temporal change of each RNA polymerase activity during sporulation. This procedure enables isolation of RNA polymerase within 4h, starting with cell pellets. Our results indicated that a principal sigma factor, sigmaA, could be detected in a holoenzyme form during all the stages of growth and sporulation, while the other sigma factors sigmaH, sigmaE, sigmaF, sigmaG, and sigmaK involved in sporulation could be detected sequentially during sporulation. Moreover, Spo0A, the central transcription factor of commitment to sporulation, was also co-purified with RNA polymerase at early stages of sporulation.
Fujita
M
M
Radioisotope Center, National Institute of Genetics, Mishima, Shizuoka 411-8540, Japan. mafujita@lab.nig.ac.jp
Sadaie
Y
Y
D001412:Bacillus subtilis / Q000166:cytology / Q000201:enzymology*; D001483:Base Sequence; D002846:Chromatography, Affinity; D002876:Chromosomes, Bacterial / Q000235:genetics; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000302:isolation & purification*; D006639:Histidine / Q000235:genetics; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification; D011995:Recombination, Genetic; D012680:Sensitivity and Specificity; D012689:Sequence Homology, Nucleic Acid; D013171:Spores, Bacterial / Q000201:enzymology; D014158:Transcription, Genetic; D014169:Transformation, Bacterial
D011993:Recombinant Fusion Proteins; D006639:Histidine; C108570:beta' subunit of RNA polymerase; D012321:DNA-Directed RNA Polymerases