Purification and functional characterization of transcription factor SII from calf thymus. Role in RNA polymerase II elongation.

ABSTRACT The coactivator complexes TRAP/SMCC and PC2 represent two forms of Mediator. To further understand the implications of the heterogeneity of the cellular Mediator populations for regulation of RNA polymerase II (Pol II) transcription, we used a combination of affinity and conventional chromatographic methods. Our analysis revealed a spectrum of complexes, including some containing significant proportions of Pol II. Interestingly, the subunit composition of the Pol II-associated Mediator population resembled that of PC2 more closely than that of the larger TRAP/SMCC complex. In in vitro transcription assays reconstituted from homogeneous preparations of general transcription factors, Mediator-associated Pol II displayed a greater specific activity (relative to that of standard Pol II) in activator-independent (basal) transcription in addition to the previously described effects of Mediator on activator-dependent transcription. Purified PC2 complex also stimulated basal activity under these conditions. Immobilized template assays in which activator-recruited preinitiation complexes were allowed to undergo one cycle of transcription revealed partial disruption of Mediator that resulted in a PC2-like complex being retained in the scaffold. This result implies that PC2 could originate as a result of a normal cellular process. Our results are thus consistent with a dynamic nature of the Mediator complex and further extend the functional similarities between Saccharomyces cerevisiae and metazoan Mediator complexes.

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Factors involved in specific transcription by mammalian RNA polymerase II: purification and characterization of general transcription factor TFIIE.

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.87.23.9163 ◽

1990 ◽

Vol 87 (23) ◽

pp. 9163-9167 ◽

Cited By ~ 51

Author(s):

Y. Ohkuma ◽

H. Sumimoto ◽

M. Horikoshi ◽

R. G. Roeder

Keyword(s):

Transcription Factor ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Purification And Characterization ◽

General Transcription Factor

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Cloning and Functional Characterization of the Gene Encoding the TFIIIB90 Subunit of RNA Polymerase III Transcription Factor TFIIIB

Journal of Biological Chemistry ◽

10.1074/jbc.271.25.14903 ◽

1996 ◽

Vol 271 (25) ◽

pp. 14903-14909 ◽

Cited By ~ 34

Author(s):

Sadia Roberts ◽

Stephen J. Miller ◽

William S. Lane ◽

Sally Lee ◽

Steven Hahn

Keyword(s):

Transcription Factor ◽

Rna Polymerase ◽

Functional Characterization ◽

Rna Polymerase Iii ◽

Gene Encoding ◽

Polymerase Iii

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Cloning and characterization of the beta subunit of human proximal sequence element-binding transcription factor and its involvement in transcription of small nuclear RNA genes by RNA polymerases II and III.

Molecular and Cellular Biology ◽

10.1128/mcb.16.10.5419 ◽

1996 ◽

Vol 16 (10) ◽

pp. 5419-5426 ◽

Cited By ~ 27

Author(s):

L Bai ◽

Z Wang ◽

J B Yoon ◽

R G Roeder

Keyword(s):

Transcription Factor ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Rna Polymerase Iii ◽

Small Nuclear Rna ◽

Class Iii ◽

Sequence Element ◽

Proximal Sequence Element ◽

Nuclear Rna

The proximal sequence element (PSE)-binding transcription factor (PTF), which binds the PSE of both RNA polymerase II- and RNA polymerase III-transcribed mammalian small nuclear RNA (snRNA) genes, is essential for their transcription. We previously reported the purification of human PTF, a complex of four subunits, and the molecular cloning and characterization of PTF gamma and delta subunits. Here we describe the isolation and expression of a cDNA encoding PTF beta, as well as functional studies using anti-PTF beta antibodies. Native PTF beta, in either protein fractions or a PTF-Oct-1-DNA complex, can be recognized by polyclonal antibodies raised against recombinant PTF beta. Immunodepletion studies show that PTF beta is required for transcription of both classes of snRNA genes in vitro. In addition, immunoprecipitation analyses demonstrate that substantial and similar molar amounts of TATA-binding protein (TBP) and TFIIIB90 can weakly associate with PTF at low salt conditions, but this association is dramatically reduced at high salt concentrations. Along with our previous demonstration of both physical interactions between PTF gamma/PTF delta and TBP and the involvement of TFIIIB90 in the transcription of class III snRNA genes, these results are consistent with the notion that a TBP-containing complex related to TFIIIB is required for the transcription of class III snRNA genes, and acts through weak interaction with the four-subunit PTF.

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