Structure and function of the DNA dependent RNA polymerase of the Archaebacterium Thermoplasma acidophilum

Abstract

DNA dependent RNA polymerase from Thermoplasma acidophilum was isolated by a procedure involving precipitation by polymin P, elution from the sediment, DEAE chromatography, heparin cellulose chromatography, sucrose glycerol gradient centrifugation and DNA cellulose chromatography. This technique has proved to be generally suitable for the isolation of RNA polymerase from Eubacteria and Archaebacteria and is probably also useful for Eukaryotes.

The purified enzyme consists of 7 components with the molecular weights 135000 108000, 56000, 35000, 22000, 13500 and 11500. The components with 56000 and 22000 daltons are absent from an incomplete inactive particle which can be separated from active enzyme by sucrose glycerol gradient centrifugation or DNA cellulose chromatography at low glycerol concentration. The component with 35000 daltons can be partially removed by DNA cellulose chromatography at high glycerol concentration and is not required for activity on poly [d(A – T) · d(A – T)]. The optimal conditions for the transcription of poly [d(A – T) · d(A–T)] and native phage DNA were determined. The activity on native phage DNA is only a few percent of that on poly [d(A – T) · d(A – T)]. It is stimulated by the addition of Mn⁺⁺ instead of Mg⁺⁺ ions and by removal of the component with the molecular weight 35000. The subunit pattern and composition are similar to those of the RNA polymerases of other Archaebacteria. The resistance against rifampicin, streptolydigine and α-amanitine is shared with all other known archaebacterial RNA polymerases.