Properties of PC4 and an RNA polymerase II complex in directing activated and basal transcription in vitro

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Abstract

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.

Original languageEnglish (US)
Pages (from-to)12492-12496
Number of pages5
JournalJournal of Biological Chemistry
Volume273
Issue number20
DOIs
StatePublished - May 15 1998

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TATA-Box Binding Protein
RNA Polymerase II
Transcription
Transcription Factor TFIID
Activator Appliances
Transcription Factor TFIIA
TATA-Binding Protein Associated Factors
General Transcription Factors
HeLa Cells
Epitopes
Cell Line
DNA
Chemical activation
Cells
In Vitro Techniques

ASJC Scopus subject areas

  • Biochemistry

Cite this

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title = "Properties of PC4 and an RNA polymerase II complex in directing activated and basal transcription in vitro",
abstract = "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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