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

Abstract

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.

Identification of autoantibodies to RNA polymerase II. Occurrence in systemic sclerosis and association with autoantibodies to RNA polymerases I and III.

“…Purification of RNA polymerase II from calf thymus was carried out by a modification ofthe method of Hodo and Blatti (28), except that all buffers contained 0.1% Nonidet P-40. Fractions from the phosphocellulose column containing RNAP II activity were pooled and applied to a heparin-agarose column equilibrated with buffer containing 60 mM ammonium sulfate.…”

Intrinsic sites of transcription termination and pausing in the c-myc gene.

“…However, analysis of specific transcription products by polyacrylamide gel electrophoresis revealed that these enzyme preparations contained a small amount of RNA polymerase III activity resistant to 2 pLg of a-amanitin per ml. (Residual RNA polymerase III has also been observed [51] in RNA polymerase II preparations purified by the method of Hodo and Blatti [27] were obtained from D. Bentley and M. Groudine. The templates used for transcription were generated by restriction digestion of the plasmid at a unique site followed by addition of single-stranded polydeoxycytidylate extensions of 30 to 100 residues by using terminal deoxynucleotidyl transferase (28).…”

Analysis of the Interaction of the Novel RNA Polymerase II (pol II) Subunit hsRPB4 with Its Partner hsRPB7 and with pol II

“…While a number of groups have purified human pol II and associated proteins (26,32,36,45,51; also reviewed in references 57 and 70), it has been clear from a comparison of results that the number, molecular weight, and quantity of copurified proteins are somewhat variable between different groups and starting sources of material, and it was previously uncertain whether a human RPB4 homolog existed. The ϳ27 kDa hsRPB7 species and ϳ18-to 19-kDa native hsRPB4 species described in this work are now shown to be present in affinity-purified pol II, conclusively demonstrating their functionality in mammalian cells.…”