TY - JOUR
T1 - Properties of PC4 and an RNA polymerase II complex in directing activated and basal transcription in vitro
AU - Wu, Shwu Yuan
AU - Chiang, Cheng Ming
PY - 1998/5/15
Y1 - 1998/5/15
N2 - 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 has no effect on the later steps of the transcriptional process.
AB - 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 has no effect on the later steps of the transcriptional process.
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U2 - 10.1074/jbc.273.20.12492
DO - 10.1074/jbc.273.20.12492
M3 - Article
C2 - 9575207
AN - SCOPUS:0032524344
SN - 0021-9258
VL - 273
SP - 12492
EP - 12496
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 20
ER -