DNA-dependent RNA polymerase from the archaebacterium Sulfolobus acidocaldarius

Abstract

Purified DNA-dependent RNA polymerase from Sulfolobus acidocaldarius is composed of 10 different subunits, one of which is present as four copies. Their molecular weights are 122 000, 101 000, 44 000, 32 000, 24 000, 17 500, 13 800, 11 800 (four copies), 11 200, 10 800, summing up to a total Mr of 423 500. The sedimentation velocity is 13.5 S, indicating that at 0.5 M NH4Cl the enzyme exists in the monomeric form. At pH 9.2 in cellogel electrophoresis two of the subunits migrate towards the cathode. The composition is quite different from that of a typical eubacterial RNA polymerase. Its complexity reminds one of eucaryotic RNA polymerase. Maximal transcription of DNA from a Halobacterium halobium phage øH (øH DNA) proceeds at pH 8.5 AND 75 DEGREES C. The enzyme is stable up to 75 degrees C and strictly requires a DNA template. øH DNA and poly[d(A-T) . d(A-T)] are the most efficient. The temperature dependence of the transcription rate is characteristic for the template. Actinomycin D and heparin prevent transcription, while rifampicin, streptolydigin and alpha-amanitin have no effect. During storage, even at -- 70 degrees C, the enzyme loses its activity to transcribe øH DNA, whereas transcription of poly[d(A-T) . D(A-6)] remains unaffected.