RNA polymerase II cofactor PC2 facilitates activation of transcription by GAL4-AH in vitro

We have isolated from a crude Hela cell cofactor fraction (USA) a novel positive cofactor that cooperates with the general transcription machinery to effect efficient stimulation of transcription by GAL4-AH, a derivative of the Saccharomyces cerevisiae regulatory factor GAL4. PC2 was shown to be a 500-kDa protein complex and to be functionally and biochemically distinct from native TFIID and previously identified cofactors. In the presence of native TFIID and other general factors, PC2 was necessary and sufficient for activation by GAL4-AH. Cofactor function was specific for transcriptional activation domains of GAL4-AH. The repressor histone H1 further potentiated but was not required for activation of transcription by GAL4-AH. On the basis of the observation that PC2 exerts entirely positive effects on transcription, we propose a model in which PC2 increases the activity of the preinitiation complex in the presence of an activator, thereby establishing a specific pathway during activation of RNA polymerase II.

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A transcriptional mediator protein that is required for activation of many RNA polymerase II promoters and is conserved from yeast to humans.

Molecular and Cellular Biology ◽

10.1128/mcb.17.8.4622 ◽

1997 ◽

Vol 17 (8) ◽

pp. 4622-4632 ◽

Cited By ~ 53

Author(s):

Y C Lee ◽

S Min ◽

B S Gim ◽

Y J Kim

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Transcriptional Activation ◽

Mediator Complex ◽

Temperature Sensitive ◽

Yeast Saccharomyces Cerevisiae ◽

Inducible Promoters ◽

Activation Of Transcription

A temperature-sensitive mutation was obtained in Med6p, a component of the mediator complex from the yeast Saccharomyces cerevisiae. The mediator complex has been shown to enable transcriptional activation in vitro. This mutation in Med6p abolished activation of transcription from four of five inducible promoters tested in vivo. There was no effect, however, on uninduced transcription, transcription of constitutively expressed genes, or transcription by RNA polymerases I and III. Mediator-RNA polymerase II complex isolated from the mutant yeast strain was temperature sensitive for transcriptional activation in a reconstituted in vitro system due to a defect in initiation complex formation. A database search revealed the existence of MED6-related genes in humans and Caenorhabditis elegans, suggesting that the role of mediator in transcriptional activation is conserved throughout the evolution.

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Region of Yeast TAF 130 Required for TFIID To Associate with Promoters

Molecular and Cellular Biology ◽

10.1128/mcb.21.4.1145-1154.2001 ◽

2001 ◽

Vol 21 (4) ◽

pp. 1145-1154 ◽

Cited By ~ 14

Author(s):

Mario Mencı́a ◽

Kevin Struhl

Keyword(s):

Rna Polymerase Ii ◽

Transcriptional Activation ◽

Thermal Inactivation ◽

Yeast Cells ◽

Activation Domains ◽

Core Promoters ◽

Transcriptional Activation Domains ◽

Tfiid Complex

ABSTRACT TFIID, a multiprotein complex comprising the TATA-binding protein (TBP) and TBP-associated factors (TAFs), associates specifically with core promoters and nucleates the assembly the RNA polymerase II transcription machinery. In yeast cells, TFIID is not generally required for transcription, although it plays an important role at many promoters. Understanding of the specific functions and physiological roles of individual TAFs within TFIID has been hampered by the fact that depletion or thermal inactivation of individual TAFs generally results in dissociation of the TFIID complex. We describe here C-terminally deleted derivatives of yeast TAF130 that assemble into normal TFIID complexes but are transcriptionally inactive in vivo. In vivo, these mutant TFIID complexes are dramatically reduced in their ability to associate with all promoters tested. In vitro, a TFIID complex containing a deleted form of TAF130 associates poorly with DNA, but it is unaffected for interacting with transcriptional activation domains. These results suggest that the C-terminal region of TAF130 is required for TFIID to associate with promoters.

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Yeast NC2 Associates with the RNA Polymerase II Preinitiation Complex and Selectively Affects Transcription In Vivo

Molecular and Cellular Biology ◽

10.1128/mcb.21.8.2736-2742.2001 ◽

2001 ◽

Vol 21 (8) ◽

pp. 2736-2742 ◽

Cited By ~ 51

Author(s):

Joseph V. Geisberg ◽

Frank C. Holstege ◽

Richard A. Young ◽

Kevin Struhl

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Transcriptional Activation ◽

Negative Regulator ◽

Preinitiation Complex ◽

Positive Role ◽

Pol Ii ◽

Basal Transcription Factors

ABSTRACT NC2 (Dr1-Drap1 or Bur6-Ydr1) has been characterized in vitro as a general negative regulator of RNA polymerase II (Pol II) transcription that interacts with TATA-binding protein (TBP) and inhibits its function. Here, we show that NC2 associates with promoters in vivo in a manner that correlates with transcriptional activity and with occupancy by basal transcription factors. NC2 rapidly associates with promoters in response to transcriptional activation, and it remains associated under conditions in which transcription is blocked after assembly of the Pol II preinitiation complex. NC2 positively and negatively affects approximately 17% of Saccharomyces cerevisiaegenes in a pattern that resembles the response to general environmental stress. Relative to TBP, NC2 occupancy is high at promoters where NC2 is positively required for normal levels of transcription. Thus, NC2 is associated with the Pol II preinitiation complex, and it can play a direct and positive role at certain promoters in vivo.

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Mutational Analysis of an RNA Polymerase II Elongation Factor in Drosophila melanogaster

Molecular and Cellular Biology ◽

10.1128/mcb.25.17.7803-7811.2005 ◽

2005 ◽

Vol 25 (17) ◽

pp. 7803-7811 ◽

Cited By ~ 10

Author(s):

Mark A. Gerber ◽

Ali Shilatifard ◽

Joel C. Eissenberg

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Cultured Cells ◽

Mutational Analysis ◽

Elongation Factor ◽

Regulatory Function ◽

Polymerase Binding ◽

Necessary And Sufficient ◽

Elongation Activity

ABSTRACT The ELL family of proteins function in vitro as elongation factors for RNA polymerase II. Deletion studies have defined domains in mammalian ELL required for transcription elongation activity and RNA polymerase binding in vitro, for transformation of cultured cells when overexpressed, and for leukemogenesis and cell proliferation as part of a leukemic fusion protein. The goal of this study was to identify domains required for chromosome targeting and viability in the unique Drosophila ELL (dELL) protein. Here, we show that an N-terminal domain of dELL is necessary and sufficient for targeting to transcriptionally active puff sites in chromatin, supporting a role for this domain in recruiting dELL to elongating RNA polymerase II. We demonstrate that a central domain of dELL is required for rapid mobilization of ELL during the heat shock response, suggesting a regulatory function for this domain. Unexpectedly, transgenic dELL in which the N-terminal chromosome binding domain is deleted can complement the recessive lethality of mutations in ELL, suggesting that Drosophila ELL has an essential activity in development distinct from its role as an RNA polymerase II elongation factor.

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Artificial Recruitment of TFIID, but Not RNA Polymerase II Holoenzyme, Activates Transcription in Mammalian Cells

Molecular and Cellular Biology ◽

10.1128/mcb.20.12.4350-4358.2000 ◽

2000 ◽

Vol 20 (12) ◽

pp. 4350-4358 ◽

Cited By ~ 31

Author(s):

David R. Dorris ◽

Kevin Struhl

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Transcriptional Activation ◽

Mammalian Cells ◽

Yeast Cells ◽

Pol Ii ◽

Activation Domains ◽

Synergistic Activation ◽

The One ◽

Rna Polymerase Ii Holoenzyme

ABSTRACT In yeast cells, transcriptional activation occurs when the RNA polymerase II (Pol II) machinery is artificially recruited to a promoter by fusing individual components of this machinery to a DNA-binding domain. Here, we show that artificial recruitment of components of the TFIID complex can activate transcription in mammalian cells. Surprisingly, artificial recruitment of TATA-binding protein (TBP) activates transiently transfected and chromosomally integrated promoters with equal efficiency, whereas artificial recruitment of TBP-associated factors activates only chromosomal reporters. In contrast, artificial recruitment of various components of the mammalian Pol II holoenzyme does not confer transcriptional activation, nor does it result in synergistic activation in combination with natural activation domains. In the one case examined in more detail, the Srb7 fusion failed to activate despite being associated with the Pol II holoenzyme and being directly recruited to the promoter. Interestingly, some acidic activation domains are less effective when the promoter is chromosomally integrated rather than transiently transfected, whereas the Sp1 glutamine-rich activation domain is more effective on integrated reporters. Thus, yeast and mammalian cells differ with respect to transcriptional activation by artificial recruitment of the Pol II holoenzyme.

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The Recruitment of theSaccharomyces cerevisiaePaf1 Complex to Active Genes Requires a Domain of Rtf1 That Directly Interacts with the Spt4-Spt5 Complex

Molecular and Cellular Biology ◽

10.1128/mcb.00270-13 ◽

2013 ◽

Vol 33 (16) ◽

pp. 3259-3273 ◽

Cited By ~ 35

Author(s):

Manasi K. Mayekar ◽

Richard G. Gardner ◽

Karen M. Arndt

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Transcription Elongation ◽

Elongation Factor ◽

Physical Interaction ◽

Repeat Domain ◽

Necessary And Sufficient ◽

Active Genes ◽

Interacting Domain

Transcription elongation factors associate with RNA polymerase II and aid its translocation through chromatin. One such factor is the conserved Paf1 complex (Paf1C), which regulates gene expression through several mechanisms, including the stimulation of cotranscriptional histone modifications. Previous studies revealed a prominent role for the Rtf1 subunit in tethering Paf1C to the RNA polymerase II elongation machinery. Here, we investigated the mechanism by which Rtf1 couples Paf1C to active chromatin. We show that a highly conserved domain of Rtf1 is necessary and sufficient for mediating a physical interaction between Rtf1 and the essential transcription elongation factor Spt5. Mutations that alter this Rtf1 domain or delete the Spt5 C-terminal repeat domain (CTR) disrupt the interaction between Rtf1 and Spt5 and release Paf1C from chromatin. When expressed in cells as the only source of Rtf1, the Spt5-interacting domain of Rtf1 can associate independently with active genes in a pattern similar to that of full-length Rtf1 and in a manner dependent on the Spt5 CTR.In vitroexperiments indicate that the interaction between the Rtf1 Spt5-interacting domain and the Spt5 CTR is direct. Collectively, our results provide molecular insight into a key attachment point between Paf1C and the RNA polymerase II elongation machinery.

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In vivo binding of NF-κB to the IκBβ promoter is insufficient for transcriptional activation

Biochemical Journal ◽

10.1042/bj20060786 ◽

2006 ◽

Vol 400 (1) ◽

pp. 115-125 ◽

Cited By ~ 7

Author(s):

Bryan D. Griffin ◽

Paul N. Moynagh

Keyword(s):

Gene Regulation ◽

Transcription Factor ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Transcriptional Activation ◽

Nuclear Factor Κb ◽

Transcriptional Initiation ◽

Il Interleukin

Despite certain structural and biochemical similarities, differences exist in the function of the NF-κB (nuclear factor κB) inhibitory proteins IκBα (inhibitory κBα) and IκBβ. The functional disparity arises in part from variance at the level of gene regulation, and in particular from the substantial induction of IκBα, but not IκBβ, gene expression post-NF-κB activation. In the present study, we probe the differential effects of IL (interleukin)-1β on induction of IκBα and perform the first characterization of the human IκBβ promoter. A consensus NF-κB-binding site, capable of binding NF-κB both in vitro and in vivo, is found in the IκBβ gene 5′ flanking region. However, the IκBβ promoter was not substantially activated by pro-inflammatory cytokines, such as IL-1β and tumour necrosis factor α, that are known to cause strong activation of NF-κB. Furthermore, in contrast with IκBα, NF-κB activation did not increase expression of endogenous IκBβ as assessed by analysis of mRNA and protein levels. Unlike κB-responsive promoters, IκBβ promoter-bound p65 inefficiently recruits RNA polymerase II, which stalls at the promoter. We present evidence that this stalling is likely due to the absence of transcription factor IIH engagement, a prerequisite for RNA polymerase II phosphorylation and transcriptional initiation. Differences in the conformation of promoter-bound NF-κB may underlie the variation in the ability to engage the basal transcriptional apparatus at the IκBβ and κB-responsive promoters. This accounts for the differential expression of IκB family members in response to NF-κB activation and furthers our understanding of the mechanisms involved in transcription factor activity and IκBβ gene regulation.

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A highly conserved domain of RNA polymerase II shares a functional element with acidic activation domains of upstream transcription factors

Molecular and Cellular Biology ◽

10.1128/mcb.14.11.7507-7516.1994 ◽

1994 ◽

Vol 14 (11) ◽

pp. 7507-7516

Author(s):

H Xiao ◽

J D Friesen ◽

J T Lis

Keyword(s):

Transcription Factors ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Transcriptional Activation ◽

Normal Function ◽

Yeast Saccharomyces Cerevisiae ◽

Activation Domains ◽

Acidic Domain ◽

General Transcription Factors ◽

Derivatives Of

We report here that the largest subunit of yeast RNA polymerase II contains an acidic domain that is similar to acidic activators of transcription. This domain includes the highly conserved homology box H. A hybrid protein containing this acidic domain fused to the DNA-binding domain of GAL4 is a potent activator of transcription in the yeast Saccharomyces cerevisiae. Interestingly, mutations that reduce the upstream activating activity of this acidic domain also abolish the normal function of RNA polymerase II. Such functional defects can be rescued by the acidic activation domains of VP16 and GAL4 when inserted into the mutant derivatives of RNA polymerase II. We further show that this acidic domain of RNA polymerase II interacts directly with two general transcription factors, the TATA-binding protein and TFIIB, and that the acidic activation domain of VP16 can compete specifically with the acidic domain of the RNA polymerase for these interactions. We discuss the implications of this finding for the mechanisms of transcriptional activation in eucaryotes.

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A highly conserved domain of RNA polymerase II shares a functional element with acidic activation domains of upstream transcription factors.

Molecular and Cellular Biology ◽

10.1128/mcb.14.11.7507 ◽

1994 ◽

Vol 14 (11) ◽

pp. 7507-7516 ◽

Cited By ~ 19

Author(s):

H Xiao ◽

J D Friesen ◽

J T Lis

Keyword(s):

Transcription Factors ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Transcriptional Activation ◽

Normal Function ◽

Yeast Saccharomyces Cerevisiae ◽

Activation Domains ◽

Acidic Domain ◽

General Transcription Factors ◽

Derivatives Of

We report here that the largest subunit of yeast RNA polymerase II contains an acidic domain that is similar to acidic activators of transcription. This domain includes the highly conserved homology box H. A hybrid protein containing this acidic domain fused to the DNA-binding domain of GAL4 is a potent activator of transcription in the yeast Saccharomyces cerevisiae. Interestingly, mutations that reduce the upstream activating activity of this acidic domain also abolish the normal function of RNA polymerase II. Such functional defects can be rescued by the acidic activation domains of VP16 and GAL4 when inserted into the mutant derivatives of RNA polymerase II. We further show that this acidic domain of RNA polymerase II interacts directly with two general transcription factors, the TATA-binding protein and TFIIB, and that the acidic activation domain of VP16 can compete specifically with the acidic domain of the RNA polymerase for these interactions. We discuss the implications of this finding for the mechanisms of transcriptional activation in eucaryotes.

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