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J. Biochem, 2003, Vol. 133, No. 1 133-138
© 2003 Japanese Biochemical Society

MOLECULAR BIOLOGY

A Rapid Purification Method for Human RNA Polymerase II by Two-Step Affinity Chromatography

Jun Hasegawa1, Masaki Endou1, Takashi Narita1, Tomoko Yamada1, Yuki Yamaguchi1,3, Tadashi Wada1 and Hiroshi Handa+,1,2

1 Graduate School of Bioscience and Biotechnology and 2 Frontier Collaborative Research Center, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama 226-8501; and 3 PRESTO, Japan Science and Technology Corporation, Yokohama 226-8503

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 Co2+ 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.

+ To whom correspondence should be addressed. Phone: +81-45-924-5872, Fax: +81-45-924-5834, E-mail: hhanda{at}bio.titech.ac.jp


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