The transcription cycle of RNA polymerase II in living cells.
The transcription cycle of RNA polymerase II in living cells.
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活细胞中 RNA 聚合酶 II 的转录周期。
DOI：
10.1083/jcb.200206019
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发表时间：
2002-12-09
影响因子：
7.8
通讯作者：
Cook, Peter R
中科院分区：
文献类型：
--
作者：
Kimura, Hiroshi;Sugaya, Kimihiko;Cook, Peter R
RNA聚合酶II转录大多数真核基因。它的催化亚基用绿色荧光蛋白标记，并在同一亚基发生突变的中国仓鼠细胞中表达;它补充了缺陷，因此具有功能。光漂白揭示了两个动力学部分的聚合酶在活细胞核：·75%的人迅速移动，但·25%的人暂时不动（缔合t1/2 ≤ 20 min）和转录活性，因为与5，6-二氯-1-β-d-呋喃核糖基苯并咪唑孵育消除了它。没有检测到不动但无活性的部分，这几乎不支持稳定全酶的存在，或启动子或核亚结构上的前起始复合物的缓慢逐步组装。放线菌素D减少了快速移动的部分，这表明参与的聚合酶停滞在插入分子，而其他启动。当野生型细胞只含有内源性酶与[3 H]尿苷孵育，新生的成绩单变得饱和与氚相似的动力学（t1/2 × 14分钟）。这些数据是一致的聚合酶是移动的的一半至六分之五的转录周期，并迅速组装成preinitiation复合物。然后，大多数表达的转录单位将花费大量时间与接合的聚合酶不相关。
RNA polymerase II transcribes most eukaryotic genes. Its catalytic subunit was tagged with green fluorescent protein and expressed in Chinese hamster cells bearing a mutation in the same subunit; it complemented the defect and so was functional. Photobleaching revealed two kinetic fractions of polymerase in living nuclei: ∼75% moved rapidly, but ∼25% was transiently immobile (association t1/2 ≈ 20 min) and transcriptionally active, as incubation with 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole eliminated it. No immobile but inactive fraction was detected, providing little support for the existence of a stable holoenzyme, or the slow stepwise assembly of a preinitiation complex on promoters or the nuclear substructure. Actinomycin D decreased the rapidly moving fraction, suggesting that engaged polymerases stall at intercalated molecules while others initiate. When wild-type cells containing only the endogenous enzyme were incubated with [3H]uridine, nascent transcripts became saturated with tritium with similar kinetics (t1/2 ≈ 14 min). These data are consistent with a polymerase being mobile for one half to five sixths of a transcription cycle, and rapid assembly into the preinitiation complex. Then, most expressed transcription units would spend significant times unassociated with engaged polymerases.