A rapid purification method for human RNA polymerase II by two-step affinity chromatography.

A rapid purification method for human RNA polymerase II by two-step affinity chromatography. Research Abstract Details 

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A rapid purification method for human RNA polymerase II by two-step affinity chromatography. Abstract Text:

Jun Hasegawa,Masaki Endou,Takashi Narita,Tomoko Yamada,Yuki Yamaguchi,Tadashi Wada,Hiroshi Handa,

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.

A rapid purification method for human RNA polymerase II by two-step affinity chromatography. Publishing Authors By Initials

J Hasegawa,M Endou,T Narita,T Yamada,Y Yamaguchi,T Wada,H Handa,

For similar investigative techniques: genetic techniques: gene transfer techniques: transfection research abstracts see: investigative techniques: genetic techniques: gene transfer techniques: transfection research

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A rapid purification method for human RNA polymerase II by two-step affinity chromatography. Journal Published:

PUBLICATION TYPE: Research Support, Non-U.S. Gov

Journal: Journal of biochemistry

VOLUME: 133

Page Numbers: 133-8

Journal Abbreviation: J. Biochem.

ISSN: 0021-924X

DAY: 19

MONTH: Jan

YEAR: 2003

A rapid purification method for human RNA polymerase II by two-step affinity chromatography. Information

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LANGUAGE: eng

NlmUniqueID: 376600

A rapid purification method for human RNA polymerase II by two-step affinity chromatography. Keywords Mesh Terms:

KEYWORDS: Transfection

MESH TERMS: metabolism

Chemical & Substance for Abstract: A rapid purification method for human RNA polymerase II by two-step affinity chromatography. Information

Substance Name: RNA Polymerase II

Registry Number: EC 2.7.7.-

Grant and Affiliation Information for A rapid purification method for human RNA polymerase II by two-step affinity chromatography.

AFFILIATION: Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama 226-8501.

Country: Japan

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MEDLINETA: J Biochem

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