Purification and partial characterization of the DNA-dependent RNA polymerase from Rickettsia prowazekii

H. F. Ding, H. H. Winkler

Research output: Contribution to journalArticle

12 Citations (Scopus)

Abstract

The DNA-dependent RNA polymerase was purified from Rickettsia prowazekii, an obligate intracellular bacterial parasite. Because of limitation of available rickettsiae, the classical methods for isolation of the enzyme from other procaryotes were modified to purify RNA polymerase from small quantities of cells (25 mg of protein). The subunit composition of the rickettsial RNA polymerase was typical of a eubacterial RNA polymerase. R. prowazekii had β' (148,OOO daltons), β (142,OOO daltons), σ (85,000 daltons), and α (34,500 daltons) subunits as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The appropriate subunits of the rickettsial RNA polymerase bound to polyclonal antisera against Escherichia coli core polymerase and E. coli σ70 subunit in Western blots (immunoblots). The enzyme activity was dependent on all four ribonucleoside triphosphates, Mg2+, and DNA template. Optimal activity occurred in the presence of 10 mM MgCl2 and 50 mM NaCl. Interestingly, in striking contrast to E. coli, approximately 74% of the rickettsial RNA polymerase activity was associated with the rickettsial cell membrane at a low salt concentration (50 mM NaCl) and dissociated from the membrane at a high salt concentration (600 mM NaCl).

Original languageEnglish (US)
Pages (from-to)5624-5630
Number of pages7
JournalJournal of Bacteriology
Volume172
Issue number10
DOIs
StatePublished - Jan 1 1990

Fingerprint

Rickettsia prowazekii
DNA-Directed RNA Polymerases
Escherichia coli
Salts
Western Blotting
Ribonucleosides
Rickettsia
Magnesium Chloride
Enzymes
Sodium Dodecyl Sulfate
Immune Sera
Polyacrylamide Gel Electrophoresis
Parasites
Cell Membrane
Membranes
DNA

ASJC Scopus subject areas

  • Microbiology
  • Molecular Biology

Cite this

Purification and partial characterization of the DNA-dependent RNA polymerase from Rickettsia prowazekii. / Ding, H. F.; Winkler, H. H.

In: Journal of Bacteriology, Vol. 172, No. 10, 01.01.1990, p. 5624-5630.

Research output: Contribution to journalArticle

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