Two dissociable subunits of yeast RNA polymerase II stimulate the initiation of transcription at a promoter in vitro.
Two dissociable subunits of yeast RNA polymerase II stimulate the initiation of transcription at a promoter in vitro.
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DOI：
10.1016/s0021-9258(18)52403-0
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发表时间：
1991-01
期刊：
影响因子：
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通讯作者：
Aled M. Edwards;C. Kane;Richard A. Young;R. Kornberg
中科院分区：
文献类型：
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作者：
Aled M. Edwards;C. Kane;Richard A. Young;R. Kornberg
从酿酒酵母菌株rpb-4中纯化缺少第四和第七大亚基的RNA聚合酶II（pol II delta 4/7），其中第四大亚基的基因缺失。pol II δ 4/7与野生型pol II（全酶）在不依赖于启动子的起始/链延伸活性（22 ℃时400-800 nmol核苷酸掺入/10 min/mg蛋白质）、链延伸速率（20-25核苷酸/s）和DNA模板中暂停位点的识别方面没有区别。与pol II全酶相反，pol II delta 4/7在体外启动子指导的转录起始中是无活性的。添加等摩尔复合物的第四和第七大亚基，纯化从pol II全酶通过离子交换色谱法在尿素的存在下，恢复启动子导向的起始活性pol II δ 4/7。转录激活蛋白Gal 4-VP 16也可以通过pol II delta 4/7从具有Gal 4结合位点的启动子引发启动子指导的起始。互补菌株rpb-4，缺乏第四大亚基，和菌株Y260-1，在最大的亚基的缺陷的提取物之间观察到。这些提取物是单独无活性的，但混合物将支持启动子定向启动。因此，第四和第七大亚基可以在聚合酶分子之间穿梭。
RNA polymerase II lacking the fourth and seventh largest subunits (pol II delta 4/7) was purified from Saccharomyces cerevisiae strain rpb-4, in which the gene for the fourth largest subunit is deleted. pol II delta 4/7 was indistinguishable from wild-type pol II (holoenzyme) in promoter-independent initiation/chain elongation activity (400-800 nmol of nucleotide incorporated/10 min/mg of protein at 22 degrees C), in rate of chain elongation (20-25 nucleotides/s), and in the recognition of pause sites in the DNA template. In contrast to pol II holoenzyme, pol II delta 4/7 was inactive in promoter-directed initiation of transcription in vitro. The addition of an equimolar complex of the fourth and seventh largest subunits, purified from pol II holoenzyme by ion-exchange chromatography in the presence of urea, restored promoter-directed initiation activity to pol II delta 4/7. The transcriptional activator protein Gal4-VP16 could also elicit promoter-directed initiation by pol II delta 4/7 from a promoter with a Gal4 binding site. Complementation was observed between extracts of strain rpb-4, lacking the fourth largest subunit, and strain Y260-1, with a defect in the largest subunit. These extracts were individually inactive, but a mixture would support promoter-directed initiation. The fourth and seventh largest subunits may, therefore, shuttle between polymerase molecules.