TY - JOUR
T1 - Purification and partial characterization of the DNA-dependent RNA polymerase from Rickettsia prowazekii
AU - Ding, H. F.
AU - Winkler, H. H.
PY - 1990
Y1 - 1990
N2 - 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).
AB - 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).
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U2 - 10.1128/jb.172.10.5624-5630.1990
DO - 10.1128/jb.172.10.5624-5630.1990
M3 - Article
C2 - 2120186
AN - SCOPUS:0024987605
SN - 0021-9193
VL - 172
SP - 5624
EP - 5630
JO - Journal of Bacteriology
JF - Journal of Bacteriology
IS - 10
ER -