DNA-Dependent RNA Polymerase from Halobacterium halobium

Abstract

DNA-dependent RNA polymerase core enzyme was isolated from H. halobium. The purification is based on the finding that the enzyme is stable in 40% wt (vol/vol) glycerol, in the presence of 0.05 M MgCl2 and involves adsorption of contaminants to DEAE-cellulose, precipitation of the complex of polymerase with DNA by streptomycin sulfate, chromatography over Biogel and affinity chromatography over heparin-Sepharose or heparin-cellulose. The enzyme consists of 4 or 5 different subunits. The composition formula was estimated as (150,000) (86,000)2 (72,000)2 (49,000)3 or 2; there may be 1 or 2 different 49,000 MW subunits. RNA synthesis requires a template. Denatured DNA is more efficient than native DNA. The transcription of native DNA is specifically stimulated by the addition of a possibly .sigma.-like factor eluted from DEAE-cellulose. The fidelity of transcription is indicated by the absolute requirement for UTP besides ATP with poly[d(A-T)] as the template.