title: A rapid purification method for human RNA polymerase II by two-step affinity chromatography.
abstract: The molecular dissection of transcription mechanisms is greatly facilitated by constructing and manipulating defined transcription systems in vitro. This approach requires highly purified transcription factors. A major enzyme participating in the transcription reaction is RNA polymerase II (RNAPII), which is composed of at least 12 subunits (RPB1-12). Due to its complex structure, it is difficult to prepare highly pure RNAPII by the conventional purification procedure. We transfected HeLa cells with a plasmid expressing RPB3 with a double FLAG-histidine tag on its amino-terminus. A high yielding clone was isolated and its extracts were subjected to immunoaffinity purification and then Co(2+) affinity chromatography. This resulted in a preparation of RNAPII complexes that consisted of all the core subunits, including the double-tagged RPB3 protein. Transcription reactions with oligo (dC)-tailed templates and transcription assays involving general transcription factors revealed that the double-tagged RNAPII complexes are active and functional in basal and activated transcription. Our method is superior to the conventionally used purification procedure in that the final preparation is markedly more pure (92% versus 40%), and the procedures are much less time-consuming. Thus, this two-step affinity purification method is an uncomplicated and effective method by which active and functional RNAPII can be prepared.
authors.affiliation: Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama 226-8501.
authors.forename: Jun
authors.initials: J
authors.lastname: Hasegawa
authors.forename: Masaki
authors.initials: M
authors.lastname: Endou
authors.forename: Takashi
authors.initials: T
authors.lastname: Narita
authors.forename: Tomoko
authors.initials: T
authors.lastname: Yamada
authors.forename: Yuki
authors.initials: Y
authors.lastname: Yamaguchi
authors.forename: Tadashi
authors.initials: T
authors.lastname: Wada
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Handa
mesh_terms: D002460:Cell Line; D002846:Chromatography, Affinity / Q000379:methods*; D006367:HeLa Cells; D006801:Humans; D021122:Protein Subunits / Q000032:analysis / Q000378:metabolism; D012319:RNA Polymerase II / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D013997:Time Factors; D014158:Transcription, Genetic; D014162:Transfection
chemical_list: D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; D012319:RNA Polymerase II