Hasegawa Jun, Endou Masaki, Narita Takashi, Yamada Tomoko, Yamaguchi Yuki, Wada Tadashi, Handa Hiroshi
Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama 226-8501.
J Biochem. 2003 Jan;133(1):133-8. doi: 10.1093/jb/mvg013.
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.
通过在体外构建和操纵特定的转录系统，极大地促进了转录机制的分子剖析。这种方法需要高度纯化的转录因子。参与转录反应的一种主要酶是RNA聚合酶II（RNAPII），它由至少12个亚基（RPB1 - 12）组成。由于其结构复杂，通过传统的纯化方法很难制备出高度纯的RNAPII。我们用一个在其氨基末端带有双FLAG - 组氨酸标签的表达RPB3的质粒转染HeLa细胞。分离出一个高产克隆，并对其提取物进行免疫亲和纯化，然后进行Co(2+)亲和层析。这得到了一种由所有核心亚基组成的RNAPII复合物制剂，包括带有双标签的RPB3蛋白。用寡聚（dC）尾模板进行的转录反应以及涉及一般转录因子的转录测定表明，带有双标签的RNAPII复合物在基础转录和激活转录中具有活性且功能正常。我们的方法优于传统使用的纯化方法，因为最终制剂的纯度明显更高（92%对40%），并且步骤耗时少得多。因此，这种两步亲和纯化方法是一种简单有效的方法，通过它可以制备出有活性且功能正常的RNAPII。
