A mammalian SRB protein associated with an RNA polymerase II holoenzyme

Chao, David M.; Gadbois, Ellen L.; Murray, Peter J.; Anderson, Stephen F.; Sonu, Michelle S.; Parvin, Jeffrey D.; Young, Richard A.

doi:10.1038/380082a0

Cited by 135 publications

(121 citation statements)

References 26 publications

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“…PCR amplifications to produce RY7133, RY7134, RY7136, RY7137, and RY7138 were performed with Vent DNA polymerase (New England Biolabs) as described by the manufacturer. The glutathione S-transferase (GST) fusions were constructed as described (13), and the ncb1⌬1 allele was constructed as described (35).…”

Section: Methodsmentioning

confidence: 99%

“…The yeast RNA polymerase II holoenzyme is a large multisubunit complex containing RNA polymerase II, a subset of the general transcription factors, and SRB regulatory proteins (7-11). Mammalian RNA polymerase II holoenzymes have also been purified, and an SRB7 homologue has been identified as a component of those complexes (12)(13)(14).For some class II genes, regulation appears to involve both positive and negative transcriptional regulators. The negative regulators that have been described include proteins purified for their ability to inhibit transcription in vitro (15-21) and genes identified because their products repress transcription from a subset of class II genes in vivo (21-29).…”

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Functional antagonism between RNA polymerase II holoenzyme and global negative regulator NC2 in vivo

Gadbois

Chao

Reese

et al. 1997

Proc. Natl. Acad. Sci. U.S.A.

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Activation of eukaryotic class II gene expression involves the formation of a transcription initiation complex that includes RNA polymerase II, general transcription factors, and SRB components of the holoenzyme. Negative regulators of transcription have been described, but it is not clear whether any are general repressors of class II genes in vivo. We reasoned that defects in truly global negative regulators should compensate for deficiencies in SRB4 because SRB4 plays a positive role in holoenzyme function. Genetic experiments reveal that this is indeed the case: a defect in the yeast homologue of the human negative regulator NC2 (Dr1⅐DRAP1) suppresses a mutation in SRB4. Global defects in mRNA synthesis caused by the defective yeast holoenzyme are alleviated by the NC2 suppressing mutation in vivo, indicating that yeast NC2 is a global negative regulator of class II transcription. These results imply that relief from repression at class II promoters is a general feature of gene activation in vivo.Activation of class II gene transcription in eukaryotes involves the recruitment of a transcription initiation complex that includes the RNA polymerase II holoenzyme (1-6). The yeast RNA polymerase II holoenzyme is a large multisubunit complex containing RNA polymerase II, a subset of the general transcription factors, and SRB regulatory proteins (7-11). Mammalian RNA polymerase II holoenzymes have also been purified, and an SRB7 homologue has been identified as a component of those complexes (12)(13)(14).For some class II genes, regulation appears to involve both positive and negative transcriptional regulators. The negative regulators that have been described include proteins purified for their ability to inhibit transcription in vitro (15-21) and genes identified because their products repress transcription from a subset of class II genes in vivo (21-29). For example, the human proteins NC1 (15, 16), NC2 or Dr1⅐DRAP1 (16,17,20), and DNA topoisomerase I (18, 19) repress basal transcription in vitro. The products of the yeast genes MOT1 (21-24), NOT1-4 (25-27), and SIN4 (28-29) negatively regulate at least a subset of yeast genes in vivo. Whether any of these negative regulators are generally employed for class II gene regulation in vivo is not yet clear.The RNA polymerase II C-terminal domain and the associated SRB complex have been implicated in the response to transcriptional activators (7-9, 30, 31). Two holoenzyme components, SRB4 and SRB6, have been shown to play essential and positive roles in transcription at the majority of class II genes in Saccharomyces cerevisiae (32). We reasoned that a defect in SRB4 might be alleviated by defects in general negative regulators and that knowledge of such regulators could contribute to our understanding of the mechanisms involved in gene regulation in vivo. Here we show that a deficiency in yeast NC2 can compensate for the global transcriptional defects caused by mutations in the SRB4 and SRB6 subunits of the RNA polymerase II holoenzyme and that NC2 is a globa...

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Section: Methodsmentioning

confidence: 99%

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confidence: 99%

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Functional antagonism between RNA polymerase II holoenzyme and global negative regulator NC2 in vivo

Gadbois

Chao

Reese

et al. 1997

Proc. Natl. Acad. Sci. U.S.A.

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“…Some of the coactivators are associated with TBP, the TATA box-binding protein, and RNA polymerase II, respectively (5)(6)(7)(8). There is also another group of coactivators that appear not to be integral parts of the basal machinery.…”

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confidence: 99%

Poly(ADP-ribose) polymerase enhances activator-dependent transcription in vitro

Meisterernst

Stelzer

Roeder

1997

Proc. Natl. Acad. Sci. U.S.A.

151

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Stimulation of class II gene transcription by activators requires the general factors and coactivators (1-4). Some of the coactivators are associated with TBP, the TATA box-binding protein, and RNA polymerase II, respectively (5-8). There is also another group of coactivators that appear not to be integral parts of the basal machinery. These soluble cofactors were identified on the basis of their capacity to enhance activation of transcription in vitro (9-12). In our initial study we had isolated a fraction from HeLa nuclear extracts, termed USA, for upstream factor stimulatory activity, that was essential for activator-dependent transcription in the presence of a complete set of general factors (9). Subsequent studies resolved the USA fraction into a minimum of four different positive cofactors, termed PC1 to PC4 (9-15). Several specific components of the USA fraction and related cofactors have been characterized in recent years. Examples include topoisomerase I, also called PC3 or Dr2 (13, 16), topoisomerase II (17), high mobility group proteins (18), and a protein called p15 or PC4 (14,15). Although PC1 and PC2 were discovered earlier, their identities are yet unresolved. Here we have identified human poly(ADP-ribose) polymerase (PARP) as one active component of the PC1 fraction.Mammalian PARP is a nuclear chromatin-associated protein of size 114 kDa that catalyzes the transfer of ADP-ribose units from NAD ϩ to nuclear protein acceptors (19)(20)(21)(22). Up to several hundred ADP-ribose units are transferred to PARP itself. Subsequently PARP modifies cellular proteins that are located within the chromatin. Target proteins include topoisomerases I and II, histones, and high mobility group proteins (23). The activity of PARP is strongly stimulated by the presence of nicks and strand breaks in DNA. These observations have contributed to the idea that PARP mediates stressinduced signaling and functions in an NAD-dependent manner in certain DNA repair processes (19,24,25). There is convincing evidence for the binding of PARP to damaged DNA containing single-strand breaks and nucleotide excisions. Automodification releases PARP from DNA, thus providing a mechanism for rendering DNA more accessible to the DNA repair machinery (23). In the absence of NAD, PARP inhibits DNA repair through binding to damaged DNA (26). Other functions proposed for PARP include roles in cellular NAD depletion (27), antirecombination and genomic stability (28), and DNA replication (29). PARP also serves as a marker for the onset of apoptosis, after which it is cleaved by proteases into DNA-binding and catalytic fragments (30).Earlier studies demonstrated that PARP suppresses nickinduced transcription in crude cell-free systems, whereas it was not required for basal transcription in systems reconstituted with purified factors (31). Moreover, inhibitors of PARP failed to demonstrate an essential role in transcription (32). However, PARP also proved to be nonessential in vivo for most of the functions suggested by the in vitro studie...

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“…The holoenzyme consists of a subset of GTFs, human SRBs (suppressors of mutations in polymerase B) which bind to the CTD and confer responsiveness to activators, and proteins involved in chromatin remodeling (SWI/SNF) and nucleotide excision repair (7,33,36,41,44,47). The holoenzyme preassembles independently of DNA, binds the promoter in a single step, and is responsive to transcriptional activators (29).…”

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The Human Immunodeficiency Virus Transactivator Tat Interacts with the RNA Polymerase II Holoenzyme

Cujec

Cho

Maldonado

et al. 1997

Molecular and Cellular Biology

108

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The human immunodeficiency virus (HIV) encodes a transcriptional transactivator (Tat), which binds to an RNA hairpin called the transactivation response element (TAR) that is located downstream of the site of initiation of viral transcription. Tat stimulates the production of full-length viral transcripts by RNA polymerase II (pol II). In this study, we demonstrate that Tat coimmunoprecipitates with the pol II holoenzyme in cells and that it binds to the purified holoenzyme in vitro. Furthermore, Tat affinity chromatography purifies a holoenzyme from HeLa nuclear extracts which, upon addition of TBP and TFIIB, supports Tat transactivation in vitro, indicating that it contains all the cellular proteins required for the function of Tat. By demonstrating that Tat interacts with the holoenzyme in the absence of TAR, our data suggest a single-step assembly of Tat and the transcription complex on the long terminal repeat of HIV.

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A mammalian SRB protein associated with an RNA polymerase II holoenzyme

Cited by 135 publications

References 26 publications

Functional antagonism between RNA polymerase II holoenzyme and global negative regulator NC2 in vivo

Functional antagonism between RNA polymerase II holoenzyme and global negative regulator NC2 in vivo

Poly(ADP-ribose) polymerase enhances activator-dependent transcription in vitro

The Human Immunodeficiency Virus Transactivator Tat Interacts with the RNA Polymerase II Holoenzyme

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