Sugaya K, Vigneron M, Cook P R
Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford, OX1 3RE, UK.
J Cell Sci. 2000 Aug;113 ( Pt 15):2679-83. doi: 10.1242/jcs.113.15.2679.
RNA polymerase II is a multi-subunit enzyme responsible for transcription of most eukaryotic genes. It associates with other complexes to form enormous multifunctional 'holoenzymes' involved in splicing and polyadenylation. We wished to study these different complexes in living cells, so we generated cell lines expressing the largest, catalytic, subunit of the polymerase tagged with the green fluorescent protein. The tagged enzyme complements a deficiency in tsTM4 cells that have a temperature-sensitive mutation in the largest subunit. Some of the tagged subunit is incorporated into engaged transcription complexes like the wild-type protein; it both resists extraction with sarkosyl and is hyperphosphorylated at its C terminus. Remarkably, subunits bearing such a tag can be incorporated into the active enzyme, despite the size and complexity of the polymerizing complex. Therefore, these cells should prove useful in the analysis of the dynamics of transcription in living cells.
RNA聚合酶II是一种多亚基酶，负责大多数真核基因的转录。它与其他复合物结合形成巨大的多功能“全酶”，参与剪接和聚腺苷酸化过程。我们希望在活细胞中研究这些不同的复合物，因此我们构建了表达用绿色荧光蛋白标记的聚合酶最大催化亚基的细胞系。标记的酶弥补了tsTM4细胞中最大亚基存在温度敏感突变的缺陷。一些标记的亚基像野生型蛋白一样被整合到参与转录的复合物中；它既抗十二烷基肌氨酸钠提取，其C末端又发生超磷酸化。值得注意的是，尽管聚合复合物的大小和复杂性，带有这种标签的亚基仍可被整合到活性酶中。因此，这些细胞在分析活细胞中转录动力学方面应会证明是有用的。
