Purification and subunit structure of deoxyribonucleic acid-dependent ribonucleic acid polymerase II from the mouse plasmacytoma, MOPC 315.
DNA-dependent RNA polymerase II was purified from the mouse plasmacytoma, MOPC 315. Soluble enzyme was obtained from a nucleoplasmic fraction and subjected to chromatography on phosphocellulose, DEAE-cellulose, and DEAE-Sephadex ion exchange resins and was subjected to sedimentation in sucrose density gradients. A chromatographically homogeneous enzyme was obtained which was purified about 25,000-fold relative to whole cell extracts and which had a specific activity (on native DNA) similar to those reported for other purified eukaryotic class II RNA polymerase preparations. Analysis of purified RNA polymerase II by polyacrylamide gel electrophoresis under nondenaturing conditions revealed three protein bands, designated II-O, II-A, and II-B in order of electrophoretic mobility. The subunit compositions of these nondenatured bands were subsequently analyzed by electrophoresis under denaturing conditions. Each enzyme II form contained subunits with molecular weights of 140,000 (II-c), 41,000 (II-d), 30,000 (II-e), 25,000 (II-f), 22,000 (II-g), 20,000 (II-h), and 16,000 (II-i). Molar ratios were unity for all subunits except subunit II-h which had a molar ratio of 2. Each enzyme form was distinguished by its highest molecular weight subunit. II-O contained subunit II-o (molecular weight 240,000), II-A contained subunit II-a (molecular weight 205,000), and II-B contained subunit II-b (molecular weight 170,000). Total molecular weights for II-O, II-A, and II-B were calculated as 554,000, 519,000, and 484,000, respectively. In addition, the number of RNA polymerase II molecules per MOPC 315 tumor cell was calculated to be about 5 times 10-4.
D000546:Amanitins; D000818:Animals; D002417:Cattle; D002460:Cell Line; D002499:Centrifugation, Density Gradient; D002852:Chromatography, Ion Exchange; D004247:DNA / Q000494:pharmacology*; D012321:DNA-Directed RNA Polymerases / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D051379:Mice; D008970:Molecular Weight; D010954:Plasmacytoma / Q000201:enzymology*; D011487:Protein Conformation; D011489:Protein Denaturation; D013950:Thymus Gland
D000546:Amanitins; D004247:DNA; D012321:DNA-Directed RNA Polymerases