Wu S Y, Chiang C M
Department of Biochemistry, University of Illinois, Urbana, Illinois 61801, USA.
J Biol Chem. 1998 May 15;273(20):12492-8. doi: 10.1074/jbc.273.20.12492.
A human RNA polymerase II (pol II) complex was isolated from a HeLa-derived cell line that conditionally expresses an epitope-tagged RPB9 subunit of human pol II. The isolated FLAG-tagged pol II complex (f:pol II) contains a subset of general transcription factors but is devoid of TFIID and TFIIA. In conjunction with TATA-binding protein (TBP) or TFIID, f:pol II is able to mediate both basal and activated transcription by Gal4-VP16 when a transcriptional coactivator PC4 is also provided. Interestingly, PC4, in the absence of a transcriptional activator, actually functions as a repressor to inhibit basal transcription. Remarkably, TBP is able to mediate activator function in this transcription system. The presence of TBP-associated factors, however, helps overcome PC4 repression and further enhance the level of activation mediated by TBP. Alleviation of PC4 repression can also be achieved by preincubation of the transcriptional components with the DNA template. Sarkosyl disruption of preinitiation complex formation further illustrates that PC4 can only inhibit transcription prior to the assembly of a functional preinitiation complex. These results suggest that PC4 represses basal transcription by preventing the assembly of a functional preinitiation complex, but it has no effect on the later steps of the transcriptional process.
从一个源自HeLa的细胞系中分离出一种人类RNA聚合酶II（pol II）复合物，该细胞系可条件性表达带有表位标签的人类pol II的RPB9亚基。分离出的带有FLAG标签的pol II复合物（f:pol II）包含一部分通用转录因子，但不含TFIID和TFIIA。当同时提供转录共激活因子PC4时，f:pol II与TATA结合蛋白（TBP）或TFIID结合，能够介导Gal4-VP16的基础转录和激活转录。有趣的是，在没有转录激活因子的情况下，PC4实际上作为一种阻遏物来抑制基础转录。值得注意的是，TBP能够在这个转录系统中介导激活因子的功能。然而，TBP相关因子的存在有助于克服PC4的阻遏作用，并进一步提高TBP介导的激活水平。通过将转录成分与DNA模板预孵育，也可以实现PC4阻遏作用的缓解。用 Sarkosyl破坏起始前复合物的形成进一步表明，PC4只能在功能性起始前复合物组装之前抑制转录。这些结果表明，PC4通过阻止功能性起始前复合物的组装来抑制基础转录，但对转录过程的后续步骤没有影响。
