Different populations of RNA polymerase II in living mammalian cells
Different populations of RNA polymerase II in living mammalian cells
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DOI：
10.1007/s10577-005-7720-1
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发表时间：
2005-01-01
影响因子：
2.6
通讯作者：
Cook, PR
中科院分区：
文献类型：
--
作者：
Hieda, M;Winstanley, H;Cook, PR
RNA聚合酶II负责大多数真核基因的转录，但是，尽管详尽的分析，很少有人知道它如何在体内转录天然模板。我们研究了活的中国仓鼠卵巢细胞中的聚合酶动力学，使用已建立的线，表达的聚合酶（RPB 1）的最大（催化）亚基标记的绿色荧光蛋白（GFP）。遗传互补显示这种标记的聚合酶是完全功能性的。荧光损失的光漂白（FLIP）揭示了存在至少三个动力学群体的标记的聚合酶：一个大的快速交换人口，一小部分耐5，6-二氯-1-β-D-呋喃核糖基苯并咪唑（DRB），但敏感的不同的转录抑制剂（即热休克），和第三部分敏感的两种抑制剂。定量免疫印迹显示最大部分是聚合酶的无活性低磷酸化形式（即IIA）。结果与第二（DRB不敏感但热休克敏感）部分结合但不接合一致，而第三（对DRB和热休克两者敏感）部分是伸长的过度磷酸化形式（即IIO）。
RNA polymerase II is responsible for transcription of most eukaryotic genes, but, despite exhaustive analysis, little is known about how it transcribes natural templates in vivo. We studied polymerase dynamics in living Chinese hamster ovary cells using an established line that expresses the largest (catalytic) subunit of the polymerase (RPB1) tagged with the green fluorescent protein (GFP). Genetic complementation has shown this tagged polymerase to be fully functional. Fluorescence loss in photobleaching (FLIP) reveals the existence of at least three kinetic populations of tagged polymerase: a large rapidly-exchanging population, a small fraction resistant to 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) but sensitive to a different inhibitor of transcription (i.e. heat shock), and a third fraction sensitive to both inhibitors. Quantitative immunoblotting shows the largest fraction to be the inactive hypophosphorylated form of the polymerase (i.e. IIA). Results are consistent with the second (DRB-insensitive but heat-shock-sensitive) fraction being bound but not engaged, while the third (sensitive to both DRB and heat shock) is the elongating hyperphosphorylated form (i.e. IIO).