Isolation, Structure, and General Properties of Yeast Ribonucleic Acid Polymerase A (or I)

作者：Jean‐Marie Buhler, André Sentenac, Pierre Fromageot · 发表于：Journal of Biological Chemistry · 年份：1974 · DOI：10.1016/s0021-9258(20)79912-6 · 被引用次数：79 · 研究领域：RNA and protein synthesis mechanisms、Fungal and yeast genetics research、DNA and Nucleic Acid Chemistry

Abstract This paper describes a method for the isolation of RNA polymerase A (or I) from Saccharomyces cerevisiae which is rapid and achieves a 2,000-fold purification. The method involves mainly a batchwise adsorption of enzyme on phosphocellulose and on DEAE-cellulose and a sedimentation on glycerol gradient. The enzyme obtained can be differentiated from RNA polymerase B (or II) by a number of criteria including electrophoretic migration on polyacrylamide gel, sensitivity to α-amanitin, subunit structure, and optimal conditions for RNA synthesis. Polyacrylamide gel electrophoresis with sodium dodecyl sulfate reveals that yeast RNA polymerase A (or I) is made up of two large subunits in equimolar amount of 190,000 and 135,000 daltons and several smaller polypeptide chains, 1 (48,000), 1 (41,000), 2 (29,000), and 2 (16,000). The RNA synthesized on native calf thymus or yeast DNA is constituted of many short chains and of a small percentage (about 10%) of very long chains which represent the bulk of the RNA. Termination factor rho from Escherichia coli inhibits 50% the transcription of native DNA.