scholarly journals Requirement of TRAP/Mediator for Both Activator-Independent and Activator-Dependent Transcription in Conjunction with TFIID-Associated TAFIIs

2002 ◽  
Vol 22 (8) ◽  
pp. 2842-2852 ◽  
Author(s):  
Hwa Jin Baek ◽  
Sohail Malik ◽  
Jun Qin ◽  
Robert G. Roeder
Keyword(s):  
Rna Polymerase Ii ◽  
Nuclear Extract ◽  
Mediator Complex ◽  
Combined Effects ◽  
Transcriptional Activators ◽  
Direct Role ◽  
Dna Templates ◽  
Hela Nuclear Extract ◽  
General Transcription Factors ◽  
General Transcription Machinery

ABSTRACT The multiprotein human TRAP/Mediator complex, which is phylogenetically related to the yeast SRB/Mediator coactivator, facilitates activation through a wide variety of transcriptional activators. However, it remains unclear how TRAP/Mediator functions in the context of other coactivators. Here we have identified a previously uncharacterized integral subunit (TRAP25) of the complex that is apparently metazoan specific. An antibody that is specific for TRAP25 allowed quantitative immunodepletion of essentially all TRAP/Mediator components from HeLa nuclear extract, without detectably affecting levels of RNA polymerase II and corresponding general transcription factors. Surprisingly, the TRAP/Mediator-depleted nuclear extract displayed severely reduced levels of both basal and activator-dependent transcription from DNA templates. Both activities were efficiently restored upon readdition of purified TRAP/Mediator. Moreover, restoration of basal and activator-dependent transcription to extracts that were simultaneously depleted of TRAP/Mediator and TFIID (TBP plus the major TAFIIs) required addition of both TBP and associated TAFIIs, as well as TRAP/Mediator. These observations indicate that TAFIIs and Mediator are jointly required for both basal and activated transcription in the context of a more physiological complement of nuclear proteins. We propose a close mechanistic linkage between these components that most likely operates at the level of combined effects on the general transcription machinery and, in addition, a direct role for Mediator in relaying activation signals to this machinery.

scholarly journals Transcriptional potentiation of the vitellogenin B1 promoter by a combination of both nucleosome assembly and transcription factors: an in vitro dissection.

1990 ◽  
Vol 10 (8) ◽  
pp. 3926-3933 ◽  
Author(s):  
B Corthésy ◽  
P Léonnard ◽  
W Wahli
Keyword(s):  
Rna Polymerase Ii ◽  
Transcriptional Activation ◽  
Nuclear Extract ◽  
Nucleosome Assembly ◽  
Dna Templates ◽  
Hela Nuclear Extract ◽  
Naked Dna ◽  
Transcription Efficiency ◽  
High Level

The Xenopus laevis vitellogenin B1 promoter was assembled into nucleosomes in an oocyte extract. Subsequent RNA polymerase II-dependent transcription from these DNA templates fully reconstituted in chromatin in a HeLa nuclear extract was increased 50-fold compared with naked DNA. Remarkably, under specific conditions, production of a high level of transcripts occurred at very low DNA (1 ng/microliter) and HeLa nuclear protein (1.6 micrograms/microliters) concentrations. When partially reconstituted templates were used, transcription efficiency was intermediate between that of fully reconstituted and naked DNA. These results implicate chromatin in the process of the transcriptional activation observed. Depletion from the oocyte assembly extract of an NF-I-like factor which binds in the promoter region upstream of the TATA box (-114 to -101) or deletion from the promoter of the region interacting with this factor reduced the transcriptional efficiency of the assembled templates by a factor of 5, but transcription of these templates was still 10 times higher than that of naked DNA. Together, these results indicate that the NF-I-like factor participates in the very efficient transcriptional potentiation of the vitellogenin B1 promoter which occurs during nucleosome assembly.

Transcriptional potentiation of the vitellogenin B1 promoter by a combination of both nucleosome assembly and transcription factors: an in vitro dissection

1990 ◽  
Vol 10 (8) ◽  
pp. 3926-3933
Author(s):  
B Corthésy ◽  
P Léonnard ◽  
W Wahli
Keyword(s):  
Rna Polymerase Ii ◽  
Transcriptional Activation ◽  
Nuclear Extract ◽  
Nucleosome Assembly ◽  
Dna Templates ◽  
Hela Nuclear Extract ◽  
Naked Dna ◽  
Transcription Efficiency ◽  
High Level

The Xenopus laevis vitellogenin B1 promoter was assembled into nucleosomes in an oocyte extract. Subsequent RNA polymerase II-dependent transcription from these DNA templates fully reconstituted in chromatin in a HeLa nuclear extract was increased 50-fold compared with naked DNA. Remarkably, under specific conditions, production of a high level of transcripts occurred at very low DNA (1 ng/microliter) and HeLa nuclear protein (1.6 micrograms/microliters) concentrations. When partially reconstituted templates were used, transcription efficiency was intermediate between that of fully reconstituted and naked DNA. These results implicate chromatin in the process of the transcriptional activation observed. Depletion from the oocyte assembly extract of an NF-I-like factor which binds in the promoter region upstream of the TATA box (-114 to -101) or deletion from the promoter of the region interacting with this factor reduced the transcriptional efficiency of the assembled templates by a factor of 5, but transcription of these templates was still 10 times higher than that of naked DNA. Together, these results indicate that the NF-I-like factor participates in the very efficient transcriptional potentiation of the vitellogenin B1 promoter which occurs during nucleosome assembly.

scholarly journals Analysis of premature termination in c-myc during transcription by RNA polymerase II in a HeLa nuclear extract.

1991 ◽  
Vol 11 (9) ◽  
pp. 4599-4615 ◽  
Author(s):  
L London ◽  
R G Keene ◽  
R Landick
Keyword(s):  
Rna Polymerase Ii ◽  
Premature Termination ◽  
Gel Filtration ◽  
Potassium Acetate ◽  
Nuclear Extract ◽  
Hela Nuclear Extract ◽  
Growth State ◽  
Nuclear Extracts ◽  
Transcription Complexes

Transcriptional regulation of the human c-myc gene, an important aspect of cellular differentiation, occurs in part at the level of transcript elongation. In vivo, transcriptional arrest, due to either pausing or termination, occurs near the junction between the first exon and first intron and varies with the growth state of the cell. We have tested the transcription of c-myc templates in HeLa nuclear extracts. We did not observe significant arrest under standard conditions, but we found that a considerable fraction of transcription complexes stopped at the c-myc TII site (just past the first exon-intron junction) when the KCl concentration was raised to 400 mM during elongation. Transcriptional arrest at TII also was observed at KCl concentrations as low as 130 mM and when potassium acetate or potassium glutamate was substituted for KCl. Under these conditions, arrest occurred at the TII site when transcription was initiated at either the c-myc P2 promoter or the adenovirus 2 major late promoter. Further, the TII sequence itself, in forward but not reverse orientation, was sufficient to stop transcription in a HeLa nuclear extract. By separating the TII RNA from active transcription complexes by using gel filtration, we found that arrest at TII at 400 mM KCl resulted in transcript release and thus true transcriptional termination. The efficiency of termination at TII depended on the growth state of the cells from which the extracts were made, suggesting that some factor or factors control premature termination in c-myc.

scholarly journals Kin28 depletion increases association of TFIID subunits Taf1 and Taf4 with promoters in Saccharomyces cerevisiae

2019 ◽  
Author(s):  
Elisabeth R. Knoll ◽  
Z. Iris Zhu ◽  
Debasish Sarkar ◽  
David Landsman ◽  
Randall H. Morse
Keyword(s):  
Transcription Factors ◽  
Rna Polymerase Ii ◽  
Mediator Complex ◽  
Gene Promoters ◽  
Initiation Complex ◽  
Pol Ii ◽  
Promoter Escape ◽  
General Transcription Factors ◽  
Promoter Occupancy ◽  
Encoding Genes

AbstractIn eukaryotes, transcription of mRNA-encoding genes by RNA polymerase II (Pol II) begins with assembly of the pre-initiation complex (PIC), comprising Pol II and the general transcription factors. Although the pathway of PIC assembly is well established, the mechanism of assembly and the dynamics of PIC components are not fully understood. For example, only recently has it been shown in yeast that the Mediator complex, which assists in pre-initiation complex formation at promoters of essentially all genes transcribed by Pol II, normally occupies promoters only transiently. This was inferred from studies showing that inhibiting Pol II promoter escape by depleting or inactivating Kin28 resulted in increased promoter occupancy by Mediator, as measured by chromatin immunoprecipitation (ChIP). Here we show that two subunits of TFIID, Taf1 and Taf4, similarly show increased occupancy as measured by ChIP upon depletion or inactivation of Kin28. In contrast, TBP occupancy is unaffected by depletion of Kin28, thus revealing an uncoupling of Taf and TBP occupancy during the transcription cycle. Increased Taf1 occupancy upon Kin28 depletion is suppressed by depletion of TBP, while depletion of TBP in the presence of Kin28 has little effect on Taf1 occupancy. Taf1 occupancy relative to TBP is higher at TFIID-dominated promoters and promoters having consensus TATA elements than at SAGA-dominated promoters and promoters lacking consensus TATA elements, consistent with prior work, and the increase in Taf occupancy upon depletion of Kin28 is more pronounced at TFIID-dominated promoters. Our results support the suggestion, based on recent structural studies, that TFIID may not remain bound to gene promoters through the transcription initiation cycle.Author SummaryTranscription of mRNA-encoding genes by RNA polymerase II (Pol II) begins when the pre-initiation complex, a large complex comprising Pol II and several general transcription factors, including the TATA-binding protein (TBP)-containing TFIID complex, assembles at gene promoters. Although the major steps in the pathway of PIC assembly have been identified, the mechanism of assembly in vivo and the dynamics of PIC components are not fully understood. In this work we have used a yeast strain that is engineered to allow inhibition of promoter escape by Pol II by administration of a chemical, in order to “freeze” the assembled PIC and thus determine whether this condition increases the promoter occupancy of TBP and two TBP-associated factors (Tafs) that are components of TFIID. This approach was used recently to demonstrate that the Mediator complex, which facilitates PIC assembly, normally binds only transiently to gene promoters. We find that Tafs, like Mediator, show increased occupancy when Pol II promoter escape is inhibited, whereas TBP binding is constant. These results imply that binding of TBP and Tafs is uncoupled during the transcription cycle, and that Taf occupancy is at least partially interrupted upon Pol II promoter escape.

scholarly journals HIV-1 Tat-associated RNA polymerase C-terminal domain kinase, CDK2, phosphorylates CDK7 and stimulates Tat-mediated transcription

2002 ◽  
Vol 364 (3) ◽  
pp. 649-657 ◽  
Author(s):  
Sergei NEKHAI ◽  
Meisheng ZHOU ◽  
Anne FERNANDEZ ◽  
William S. LANE ◽  
Ned J.C. LAMB ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Nuclear Extract ◽  
Viral Gene ◽  
Transcriptional Elongation ◽  
Rna Pol Ii ◽  
Hela Nuclear Extract ◽  
Pol Ii ◽  
Terminal Domain ◽  
Hiv 1

HIV-1 gene expression is regulated by a viral transactivator protein (Tat) which induces transcriptional elongation of HIV-1 long tandem repeat (LTR). This induction requires hyperphosphorylation of the C-terminal domain (CTD) repeats of RNA polymerase II (Pol II). To achieve CTD hyperphosphorylation, Tat stimulates CTD kinases associated with general transcription factors of the promoter complex, specifically TFIIH-associated CDK7 and positive transcription factor b-associated CDK9 (cyclin-dependent kinase 9). Other studies indicate that Tat may bind an additional CTD kinase that regulates the target-specific phosphorylation of RNA Pol II CTD. We previously reported that Tat-associated T-cell-derived kinase (TTK), purified from human primary T-cells, stimulates Tat-dependent transcription of HIV-1 LTR in vivo [Nekhai, Shukla, Fernandez, Kumar and Lamb (2000) Virology 266, 246–256]. In the work presented here, we characterized the components of TTK by biochemical fractionation and the function of TTK in transcription assays in vitro. TTK uniquely co-purified with CDK2 and not with either CDK9 or CDK7. Tat induced the TTK-associated CDK2 kinase to phosphorylate CTD, specifically at Ser-2 residues. The TTK fraction restored Tat-mediated transcription activation of HIV-1 LTR in a HeLa nuclear extract immunodepleted of CDK9, but not in the HeLa nuclear extract double-depleted of CDK9 and CDK7. Direct microinjection of the TTK fraction augmented Tat transactivation of HIV-1 LTR in human primary HS68 fibroblasts. The results argue that TTK-associated CDK2 may function to maintain target-specific phosphorylation of RNA Pol II that is essential for Tat transactivation of HIV-1 promoter. They are also consistent with the observed cell-cycle-specific induction of viral gene transactivation.

scholarly journals Native and Recombinant Polycomb Group Complexes Establish a Selective Block to Template Accessibility To Repress Transcription In Vitro

2002 ◽  
Vol 22 (22) ◽  
pp. 7919-7928 ◽  
Author(s):  
Ian F. G. King ◽  
Nicole J. Francis ◽  
Robert E. Kingston
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Nuclear Extract ◽  
Polycomb Group ◽  
Core Complex ◽  
In Vitro System ◽  
Hela Nuclear Extract ◽  
Transcription In Vitro ◽  
Polycomb Group Complexes

ABSTRACT Polycomb group (PcG) proteins are responsible for stable repression of homeotic gene expression during Drosophila melanogaster development. They are thought to stabilize chromatin structure to prevent transcription, though how they do this is unknown. We have established an in vitro system in which the PcG complex PRC1 and a recombinant PRC1 core complex (PCC) containing only PcG proteins are able to repress transcription by both RNA polymerase II and by T7 RNA polymerase. We find that assembly of the template into nucleosomes enhances repression by PRC1 and PCC. The subunit Psc is able to inhibit transcription on its own. PRC1- and PCC-repressed templates remain accessible to Gal4-VP16 binding, and incubation of the template with HeLa nuclear extract before the addition of PCC eliminates PCC repression. These results suggest that PcG proteins do not merely prohibit all transcription machinery from binding the template but instead likely inhibit specific steps in the transcription reaction.

scholarly journals Yeast Cap Binding Complex Impedes Recruitment of Cleavage Factor IA to Weak Termination Sites

2007 ◽  
Vol 27 (18) ◽  
pp. 6520-6531 ◽  
Author(s):  
Chi-Ming Wong ◽  
Hongfang Qiu ◽  
Cuihua Hu ◽  
Jinsheng Dong ◽  
Alan G. Hinnebusch
Keyword(s):  
Rna Polymerase Ii ◽  
Mutant Allele ◽  
Mrna Splicing ◽  
Transcriptional Activators ◽  
Direct Role ◽  
Pol Ii ◽  
Naturally Occurring ◽  
Cap Binding Complex ◽  
A Site ◽  
Nascent Transcripts

ABSTRACT Nuclear cap binding complex (CBC) is recruited cotranscriptionally and stimulates spliceosome assembly on nascent mRNAs; however, its possible functions in regulating transcription elongation or termination were not well understood. We show that, while CBC appears to be dispensable for normal rates and processivity of elongation by RNA polymerase II (Pol II), it plays a direct role in preventing polyadenylation at weak termination sites. Similarly to Npl3p, with which it interacts, CBC suppresses the weak terminator of the gal10-Δ56 mutant allele by impeding recruitment of termination factors Pcf11p and Rna15p (subunits of cleavage factor IA [CF IA]) and does so without influencing Npl3p occupancy at the termination site. Importantly, deletion of CBC subunits or NPL3 also increases termination at a naturally occurring weak poly(A) site in the RNA14 coding sequences. We also show that CBC is most likely recruited directly to the cap of nascent transcripts rather than interacting first with transcriptional activators or the phosphorylated C-terminal domain of Pol II. Thus, our findings illuminate the mechanism of CBC recruitment and extend its function in Saccharomyces cerevisiae beyond mRNA splicing and degradation of aberrant nuclear mRNAs to include regulation of CF IA recruitment at poly(A) selection sites.

scholarly journals Kin28 depletion increases association of TFIID subunits Taf1 and Taf4 with promoters in Saccharomyces cerevisiae

2020 ◽  
Vol 48 (8) ◽  
pp. 4244-4255 ◽  
Author(s):  
Elisabeth R Knoll ◽  
Z Iris Zhu ◽  
Debasish Sarkar ◽  
David Landsman ◽  
Randall H Morse
Keyword(s):  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Mediator Complex ◽  
Gene Promoters ◽  
Structural Studies ◽  
Initiation Complex ◽  
Pol Ii ◽  
Promoter Escape ◽  
General Transcription Factors ◽  
Encoding Genes

Abstract Transcription of eukaryotic mRNA-encoding genes by RNA polymerase II (Pol II) begins with assembly of the pre-initiation complex (PIC), comprising Pol II and the general transcription factors. Although the pathway of PIC assembly is well established, the mechanism of assembly and the dynamics of PIC components are not fully understood. For example, only recently has it been shown that in yeast, the Mediator complex normally occupies promoters only transiently, but shows increased association when Pol II promoter escape is inhibited. Here we show that two subunits of TFIID, Taf1 and Taf4, similarly show increased occupancy as measured by ChIP upon depletion or inactivation of Kin28. In contrast, TBP occupancy is unaffected by depletion of Kin28, thus revealing an uncoupling of Taf and TBP occupancy during the transcription cycle. Increased Taf1 occupancy upon Kin28 depletion is suppressed by depletion of TBP, while depletion of TBP in the presence of Kin28 has little effect on Taf1 occupancy. The increase in Taf occupancy upon depletion of Kin28 is more pronounced at TFIID-dominated promoters compared to SAGA-dominated promoters. Our results support the suggestion, based on recent structural studies, that TFIID may not remain bound to gene promoters through the transcription initiation cycle.

Analysis of premature termination in c-myc during transcription by RNA polymerase II in a HeLa nuclear extract

1991 ◽  
Vol 11 (9) ◽  
pp. 4599-4615
Author(s):  
L London ◽  
R G Keene ◽  
R Landick
Keyword(s):  
Rna Polymerase Ii ◽  
Premature Termination ◽  
Gel Filtration ◽  
Potassium Acetate ◽  
Nuclear Extract ◽  
Hela Nuclear Extract ◽  
Growth State ◽  
Nuclear Extracts ◽  
Transcription Complexes

Transcriptional regulation of the human c-myc gene, an important aspect of cellular differentiation, occurs in part at the level of transcript elongation. In vivo, transcriptional arrest, due to either pausing or termination, occurs near the junction between the first exon and first intron and varies with the growth state of the cell. We have tested the transcription of c-myc templates in HeLa nuclear extracts. We did not observe significant arrest under standard conditions, but we found that a considerable fraction of transcription complexes stopped at the c-myc TII site (just past the first exon-intron junction) when the KCl concentration was raised to 400 mM during elongation. Transcriptional arrest at TII also was observed at KCl concentrations as low as 130 mM and when potassium acetate or potassium glutamate was substituted for KCl. Under these conditions, arrest occurred at the TII site when transcription was initiated at either the c-myc P2 promoter or the adenovirus 2 major late promoter. Further, the TII sequence itself, in forward but not reverse orientation, was sufficient to stop transcription in a HeLa nuclear extract. By separating the TII RNA from active transcription complexes by using gel filtration, we found that arrest at TII at 400 mM KCl resulted in transcript release and thus true transcriptional termination. The efficiency of termination at TII depended on the growth state of the cells from which the extracts were made, suggesting that some factor or factors control premature termination in c-myc.

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