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

1997 ◽  
Vol 17 (8) ◽  
pp. 4622-4632 ◽  
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.

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

1994 ◽  
Vol 14 (6) ◽  
pp. 3927-3937
Author(s):  
M Kretzschmar ◽  
G Stelzer ◽  
R G Roeder ◽  
M Meisterernst
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcriptional Activation ◽  
Activation Domains ◽  
Positive Effects ◽  
Activation Of Transcription ◽  
Transcriptional Activation Domains ◽  
Necessary And Sufficient ◽  
Specific Pathway

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.

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

2001 ◽  
Vol 21 (8) ◽  
pp. 2736-2742 ◽  
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.

scholarly journals Identifying a species-specific region of yeast TF11B in vivo.

1996 ◽  
Vol 16 (7) ◽  
pp. 3651-3657 ◽  
Author(s):  
S P Shaw ◽  
J Wingfield ◽  
M J Dorsey ◽  
J Ma
Keyword(s):  
Amino Acids ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcriptional Activation ◽  
Specific Region ◽  
Site Directed Mutagenesis ◽  
Yeast Cells ◽  
Yeast Saccharomyces Cerevisiae ◽  
Species Specific

The general transcription factor IIB (TFIIB) is required for RNA polymerase II transcription in eukaryotes. It provides a physical link between the TATA-binding protein (TBP) and the RNA polymerase and is a component previously suggested to respond to transcriptional activators in vitro. In this report, we compare the yeast (Saccharomyces cerevisiae) and human forms of the protein in yeast cells to study their functional differences. We demonstrate that human TFIIB fails to functionally replace yeast TFIIB in yeast cells. By analyzing various human-yeast hybrid TFIIB molecules, we show that a 14-amino-acid region at the amino terminus of the first repeat of yeast TFIIB plays an important role in determining species specificity in vivo. In addition, we identify four amino acids in this region that are critical for an amphipathic helix unique to yeast TFIIB. By site-directed mutagenesis analyses we demonstrate that these four amino acids are important for yeast TFIIB's activity in vivo. Finally, we show that mutations in the species-specific region of yeast TFIIB can differentially affect the expression of genes activated by different activators in vivo. These results provide strong evidence suggesting that yeast TFIIB is involved in the process of transcriptional activation in living cells.

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

2006 ◽  
Vol 400 (1) ◽  
pp. 115-125 ◽  
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.

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

1994 ◽  
Vol 14 (6) ◽  
pp. 3927-3937 ◽  
Author(s):  
M Kretzschmar ◽  
G Stelzer ◽  
R G Roeder ◽  
M Meisterernst
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcriptional Activation ◽  
Activation Domains ◽  
Positive Effects ◽  
Activation Of Transcription ◽  
Transcriptional Activation Domains ◽  
Necessary And Sufficient ◽  
Specific Pathway

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.

scholarly journals Multiple Mechanisms of Suppression Circumvent Transcription Defects in an RNA Polymerase Mutant

2000 ◽  
Vol 20 (21) ◽  
pp. 8124-8133 ◽  
Author(s):  
Qian Tan ◽  
Xin Li ◽  
Parag P. Sadhale ◽  
Takenori Miyao ◽  
Nancy A. Woychik
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Suppressor Cells ◽  
Suppressor Gene ◽  
Temperature Sensitive ◽  
Mrna Levels ◽  
La Protein ◽  
Significant Amelioration

ABSTRACT Using a high-copy-number suppressor screen to obtain clues about the role of the yeast RNA polymerase II subunit RPB4 in transcription, we identified three suppressors of the temperature sensitivity resulting from deletion of the RPB4 gene (ΔRPB4). One suppressor is Sro9p, a protein related to La protein, another is the nucleosporin Nsp1p, and the third is the RNA polymerase II subunit RPB7. Suppression by RPB7 was anticipated since its interaction with RPB4 is well established both in vitro and in vivo. We examined the effect of overexpression of each suppressor gene on transcription. Interestingly, suppression of the temperature-sensitive phenotype correlates with the correction of a characteristic transcription defect of this mutant: each suppressor restored the level of promoter-specific, basal transcription to wild-type levels. Examination of the effects of the suppressors on other in vivo transcription aberrations in ΔRPB4 cells revealed significant amelioration of defects in certain inducible genes in Sro9p and RPB7, but not in Nsp1p, suppressor cells. Analysis of mRNA levels demonstrated that overexpression of each of the three suppressors minimally doubled the mRNA levels during stationary phase. However, the elevated mRNA levels in Sro9p suppressor cells appear to result from a combination of enhanced transcription and message stability. Taken together, these results demonstrate that these three proteins influence transcription and implicate Sro9p in both transcription and posttranscription events.

scholarly journals Functional dissection of the catalytic mechanism of mammalian RNA polymerase II

2005 ◽  
Vol 83 (4) ◽  
pp. 497-504 ◽  
Author(s):  
Benoit Coulombe ◽  
Marie-France Langelier
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Catalytic Mechanism ◽  
Mutational Analysis ◽  
Structural Elements ◽  
Catalytic Mechanisms ◽  
Rnap Ii ◽  
Affinity Peptide

High resolution X-ray crystal structures of multisubunit RNA polymerases (RNAP) have contributed to our understanding of transcriptional mechanisms. They also provided a powerful guide for the design of experiments aimed at further characterizing the molecular stages of the transcription reaction. Our laboratory used tandem-affinity peptide purification in native conditions to isolate human RNAP II variants that had site-specific mutations in structural elements located strategically within the enzyme's catalytic center. Both in vitro and in vivo analyses of these mutants revealed novel features of the catalytic mechanisms involving this enzyme.Key words: RNA polymerase II, transcriptional mechanisms, mutational analysis, mRNA synthesis.

scholarly journals The Essential WD Repeat Protein Swd2 Has Dual Functions in RNA Polymerase II Transcription Termination and Lysine 4 Methylation of Histone H3

2004 ◽  
Vol 24 (7) ◽  
pp. 2932-2943 ◽  
Author(s):  
Hailing Cheng ◽  
Xiaoyuan He ◽  
Claire Moore
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Chromatin Modification ◽  
Histone H3 ◽  
Temperature Sensitive ◽  
Repeat Protein ◽  
Tightly Coupled ◽  
Cleavage And Polyadenylation ◽  
Wd Repeat

ABSTRACT Swd2, an essential WD repeat protein in Saccharomyces cerevisiae, is a component of two very different complexes: the cleavage and polyadenylation factor CPF and the Set1 methylase, which modifies lysine 4 of histone H3 (H3-K4). It was not known if Swd2 is important for the function of either of these entities. We show here that, in extract from cells depleted of Swd2, cleavage and polyadenylation of the mRNA precursor in vitro are completely normal. However, temperature-sensitive mutations or depletion of Swd2 causes termination defects in some genes transcribed by RNA polymerase II. Overexpression of Ref2, a protein previously implicated in snoRNA 3′ end formation and Swd2 recruitment to CPF, can rescue the growth and termination defects, indicating a functional interaction between the two proteins. Some swd2 mutations also significantly decrease global H3-K4 methylation and cause other phenotypes associated with loss of this chromatin modification, such as loss of telomere silencing, hydroxyurea sensitivity, and alterations in repression of INO1 transcription. Even though the two Swd2-containing complexes are both localized to actively transcribed genes, the allele specificities of swd2 defects suggest that the functions of Swd2 in mediating RNA polymerase II termination and H3-K4 methylation are not tightly coupled.

scholarly journals Direct Interaction between the Subunit RAP30 of Transcription Factor IIF (TFIIF) and RNA Polymerase Subunit 5, Which Contributes to the Association between TFIIF and RNA Polymerase II

2001 ◽  
Vol 276 (15) ◽  
pp. 12266-12273 ◽  
Author(s):  
Wenxiang Wei ◽  
Dorjbal Dorjsuren ◽  
Yong Lin ◽  
Weiping Qin ◽  
Takahiro Nomura ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Middle Part ◽  
Wild Type ◽  
Binding Region ◽  
Pol Ii ◽  
Transcription Factor Iif ◽  
B Virus

The general transcription factor IIF (TFIIF) assembled in the initiation complex, and RAP30 of TFIIF, have been shown to associate with RNA polymerase II (pol II), although it remains unclear which pol II subunit is responsible for the interaction. We examined whether TFIIF interacts with RNA polymerase II subunit 5 (RPB5), the exposed domain of which binds transcriptional regulatory factors such as hepatitis B virus X protein and a novel regulatory protein, RPB5-mediating protein. The results demonstrated that RPB5 directly binds RAP30in vitrousing purified recombinant proteins andin vivoin COS1 cells transiently expressing recombinant RAP30 and RPB5. The RAP30-binding region was mapped to the central region (amino acids (aa) 47–120) of RPB5, which partly overlaps the hepatitis B virus X protein-binding region. Although the middle part (aa 101–170) and the N-terminus (aa 1–100) of RAP30 independently bound RPB5, the latter was not involved in the RPB5 binding when RAP30 was present in TFIIF complex. Scanning of the middle part of RAP30 by clustered alanine substitutions and then point alanine substitutions pinpointed two residues critical for the RPB5 binding inin vitroandin vivoassays. Wild type but not mutants Y124A and Q131A of RAP30 coexpressed with FLAG-RAP74 efficiently recovered endogenous RPB5 to the FLAG-RAP74-bound anti-FLAG M2 resin. The recovered endogenous RPB5 is assembled in pol II as demonstrated immunologically. Interestingly, coexpression of the central region of RPB5 and wild type RAP30 inhibited recovery of endogenous pol II to the FLAG-RAP74-bound M2 resin, strongly suggesting that the RAP30-binding region of RPB5 inhibited the association of TFIIF and pol II. The exposed domain of RPB5 interacts with RAP30 of TFIIF and is important for the association between pol II and TFIIF.

Identification of rpo30, a vaccinia virus RNA polymerase gene with structural similarity to a eucaryotic transcription elongation factor

1990 ◽  
Vol 10 (10) ◽  
pp. 5433-5441
Author(s):  
B Y Ahn ◽  
P D Gershon ◽  
E V Jones ◽  
B Moss
Keyword(s):  
Rna Polymerase ◽  
Vaccinia Virus ◽  
Rna Polymerase Ii ◽  
Elongation Factor ◽  
Viral Rna ◽  
In Vitro Translation ◽  
Virus Protein ◽  
Transcriptional Elongation

Eucaryotic transcription factors that stimulate RNA polymerase II by increasing the efficiency of elongation of specifically or randomly initiated RNA chains have been isolated and characterized. We have identified a 30-kilodalton (kDa) vaccinia virus-encoded protein with apparent homology to SII, a 34-kDa mammalian transcriptional elongation factor. In addition to amino acid sequence similarities, both proteins contain C-terminal putative zinc finger domains. Identification of the gene, rpo30, encoding the vaccinia virus protein was achieved by using antibody to the purified viral RNA polymerase for immunoprecipitation of the in vitro translation products of in vivo-synthesized early mRNA selected by hybridization to cloned DNA fragments of the viral genome. Western immunoblot analysis using antiserum made to the vaccinia rpo30 protein expressed in bacteria indicated that the 30-kDa protein remains associated with highly purified viral RNA polymerase. Thus, the vaccinia virus protein, unlike its eucaryotic homolog, is an integral RNA polymerase subunit rather than a readily separable transcription factor. Further studies showed that the expression of rpo30 is regulated by dual early and later promoters.

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