Purification using polyethylenimine precipitation and low molecular weight subunit analyses of calf thymus and wheat germ DNA-dependent RNA polymerase II

DNA-dependent RNA polymerase II from calf thymus has been successfully purified using polythylenimine precipitation. Thus, 5-6 mg of nearly homogeneous homogeneous trna polymerase II (greater than 96% pure) can be prepared from 1 kg of calf thymus with three chromatography steps following extraction and precipitation of the enzyme from the polyethylenimine pellet. This procedure eliminates the high salt extraction of chromatin previously used in purification of this enzyme and makes possible the large scale preparation of mammalian RNA polymerase II. Calf thymus polymerase II prepared by this method is greater than 90% form IIb and consists of ten different subunits having the following molecular weights: 180 000; 145 000; 36 000; 25 000; 20 000; 18 500; 16 000; 15 000; 12 000; 11 500. The homologous enzyme isolated from wheat germ is greater than 90% form IIa and contains subunits of the following molecular weights: 206 000; 145 000; 44 000-47 000; 24 500; 21 000; 19 000; 17 000; 14 000; 13 500. The wheat germ and calf thymus enzymes exhibit similar subunits structures, but the molecular weights of individual subunits are clearly different between the enzymes. Wheat germ RNA polymerase II is 50% inhibited by 0.271 microng/mL of alpha-amanitin, a level 30-fold higher than that found for calf thymus RNA polymerase II. These enzymes are further distinguished by the absence of antigenic cross reactivity.     

Comparative study of calf thymus and wheat germ RNA polymerase II: stability of initiation complexes and elongation rates.

Pure wheat germ RNA polymerase II but not calf thymus RNA polymerase II forms relatively stable binary complexes (half life time of 30 minutes at 0°C) with superhelical SV 40 DNA. On the contrary, the addition of a specific dinucleotide and a single ribotriphosphate permits the formation of highly stable complexes between both enzymes and SV 40 DNA. The elongation of RNA chains with preinitiated wheat germ enzyme only is stimulated by sarkosyl. These observations suggest that the wheat germ enzyme, as compared to that isolated from calf thymus, may contain a protein factor, a more native structure or both that permit efficient initiation and elongation of RNA chains on double stranded DNA.