Rapid isolation of RNA polymerase from sporulating cells of Bacillus subtilis

Gene. 1998 Oct 23;221(2):185-90. doi: 10.1016/s0378-1119(98)00452-1.

Abstract

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.

MeSH terms

  • Bacillus subtilis / cytology
  • Bacillus subtilis / enzymology*
  • Base Sequence
  • Chromatography, Affinity
  • Chromosomes, Bacterial / genetics
  • DNA-Directed RNA Polymerases / genetics
  • DNA-Directed RNA Polymerases / isolation & purification*
  • Histidine / genetics
  • Molecular Sequence Data
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / isolation & purification
  • Recombination, Genetic
  • Sensitivity and Specificity
  • Sequence Homology, Nucleic Acid
  • Spores, Bacterial / enzymology
  • Transcription, Genetic
  • Transformation, Bacterial

Substances

  • Recombinant Fusion Proteins
  • Histidine
  • beta' subunit of RNA polymerase
  • DNA-Directed RNA Polymerases