A rapid purification method for human RNA polymerase II by two-step affinity chromatography.
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.
Hasegawa
Jun
J
Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama 226-8501.
Endou
Masaki
M
Narita
Takashi
T
Yamada
Tomoko
T
Yamaguchi
Yuki
Y
Wada
Tadashi
T
Handa
Hiroshi
H
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
D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; D012319:RNA Polymerase II