scholarly journals TATA-Binding Protein Mutants That Are Lethal in the Absence of the Nhp6 High-Mobility-Group Protein

2004 ◽  
Vol 24 (14) ◽  
pp. 6419-6429 ◽  
Author(s):  
Peter Eriksson ◽  
Debabrata Biswas ◽  
Yaxin Yu ◽  
James M. Stewart ◽  
David J. Stillman
Keyword(s):  
Rna Polymerase Ii ◽  
Transcriptional Activation ◽  
Synthetic Lethality ◽  
Binding Protein ◽  
High Mobility ◽  
Tata Binding Protein ◽  
High Mobility Group ◽  
Multicopy Plasmid ◽  
Pol Ii ◽  
Pol Iii

ABSTRACT The Saccharomyces cerevisiae Nhp6 protein is related to the high-mobility-group B family of architectural DNA-binding proteins that bind DNA nonspecifically but bend DNA sharply. Nhp6 is involved in transcriptional activation by both RNA polymerase II (Pol II) and Pol III. Our previous genetic studies have implicated Nhp6 in facilitating TATA-binding protein (TBP) binding to some Pol II promoters in vivo, and we have used a novel genetic screen to isolate 32 new mutations in TBP that are viable in wild-type cells but lethal in the absence of Nhp6. The TBP mutations that are lethal in the absence of Nhp6 cluster in three regions: on the upper surface of TBP that may have a regulatory role, near residues that contact Spt3, or near residues known to contact either TFIIA or Brf1 (in TFIIIB). The latter set of mutations suggests that Nhp6 becomes essential when a TBP mutant compromises its ability to interact with either TFIIA or Brf1. Importantly, the synthetic lethality for some of the TBP mutations is suppressed by a multicopy plasmid with SNR6 or by an spt3 mutation. It has been previously shown that nhp6ab mutants are defective in expressing SNR6, a Pol III-transcribed gene encoding the U6 splicing RNA. Chromatin immunoprecipitation experiments show that TBP binding to SNR6 is reduced in an nhp6ab mutant. Nhp6 interacts with Spt16/Pob3, the yeast equivalent of the FACT elongation complex, consistent with nhp6ab cells being extremely sensitive to 6-azauracil (6-AU). However, this 6-AU sensitivity can be suppressed by multicopy SNR6 or BRF1. Additionally, strains with SNR6 promoter mutations are sensitive to 6-AU, suggesting that decreased SNR6 RNA levels contribute to 6-AU sensitivity. These results challenge the widely held belief that 6-AU sensitivity results from a defect in transcriptional elongation.

scholarly journals Regulation of TATA-Binding Protein Binding by the SAGA Complex and the Nhp6 High-Mobility Group Protein

2003 ◽  
Vol 23 (6) ◽  
pp. 1910-1921 ◽  
Author(s):  
Yaxin Yu ◽  
Peter Eriksson ◽  
Leena T. Bhoite ◽  
David J. Stillman
Keyword(s):  
Transcription Factor ◽  
Synthetic Lethality ◽  
Binding Protein ◽  
High Mobility ◽  
Tata Binding Protein ◽  
High Mobility Group ◽  
Saga Complex ◽  
High Mobility Group Protein ◽  
Group Protein

ABSTRACT Transcriptional activation of the yeast HO gene involves the sequential action of DNA-binding and chromatin-modifying factors. Here we examine the role of the SAGA complex and the Nhp6 architectural transcription factor in HO regulation. Our data suggest that these factors regulate binding of the TATA-binding protein (TBP) to the promoter. A gcn5 mutation, eliminating the histone acetyltransferase present in SAGA, reduces the transcription of HO, but expression is restored in a gcn5 spt3 double mutant. We conclude that the major role of Gcn5 in HO activation is to overcome repression by Spt3. Spt3 is also part of SAGA, and thus two proteins in the same regulatory complex can have opposing roles in transcriptional regulation. Chromatin immunoprecipitation experiments show that TBP binding to HO is very weak in wild-type cells but markedly increased in an spt3 mutant, indicating that Spt3 reduces HO expression by inhibiting TBP binding. In contrast, it has been shown previously that Spt3 stimulates TBP binding to the GAL1 promoter as well as GAL1 expression, and thus, Spt3 regulates these promoters differently. We also find genetic interactions between TBP and either Gcn5 or the high-mobility-group protein Nhp6, including multicopy suppression and synthetic lethality. These results suggest that, while Spt3 acts to inhibit TBP interaction with the HO promoter, Gcn5 and Nhp6 act to promote TBP binding. The result of these interactions is to limit TBP binding and HO expression to a short period within the cell cycle. Furthermore, the synthetic lethality resulting from combining a gcn5 mutation with specific TBP point mutations can be suppressed by the overexpression of transcription factor IIA (TFIIA), suggesting that histone acetylation by Gcn5 can stimulate transcription by promoting the formation of a TBP/TFIIA complex.

scholarly journals An Array of Coactivators Is Required for Optimal Recruitment of TATA Binding Protein and RNA Polymerase II by Promoter-Bound Gcn4p

2004 ◽  
Vol 24 (10) ◽  
pp. 4104-4117 ◽  
Author(s):  
Hongfang Qiu ◽  
Cuihua Hu ◽  
Sungpil Yoon ◽  
Krishnamurthy Natarajan ◽  
Mark J. Swanson ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcriptional Activation ◽  
Target Genes ◽  
Binding Protein ◽  
Tata Binding Protein ◽  
Pol Ii ◽  
Elongation Phase ◽  
Target Promoters

ABSTRACT Wild-type transcriptional activation by Gcn4p is dependent on multiple coactivators, including SAGA, SWI/SNF, Srb mediator, CCR4-NOT, and RSC, which are all recruited by Gcn4p to its target promoters in vivo. It was not known whether these coactivators are required for assembly of the preinitiation complex (PIC) or for subsequent steps in the initiation or elongation phase of transcription. We find that mutations in subunits of these coactivators reduce the recruitment of TATA binding protein (TBP) and RNA polymerase II (Pol II) by Gcn4p at ARG1, ARG4, and SNZ1, implicating all five coactivators in PIC assembly at Gcn4p target genes. Recruitment of Pol II at SNZ1 and ARG1 was eliminated by mutations in TBP or by deletion of the TATA box, indicating that TBP binding is a prerequisite for Pol II recruitment by Gcn4p. However, several mutations in SAGA subunits and deletion of SRB10 had a greater impact on promoter occupancy of Pol II versus TBP, suggesting that SAGA and Srb mediator can promote Pol II binding independently of their stimulatory effects on TBP recruitment. Our results reveal an unexpected complexity in the cofactor requirements for the enhancement of PIC assembly by a single activator protein.

scholarly journals MAF1 represses CDKN1A through a Pol III-dependent mechanism

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Yu-Ling Lee ◽  
Yuan-Ching Li ◽  
Chia-Hsin Su ◽  
Chun-Hui Chiao ◽  
I-Hsuan Lin ◽  
...  
Keyword(s):  
Chromatin Immunoprecipitation ◽  
Binding Protein ◽  
Tata Binding Protein ◽  
Protein Coding ◽  
Pol Ii ◽  
Histone Marks ◽  
Chromatin Looping ◽  
Pol Iii ◽  
Dependent Mechanism

MAF1 represses Pol III-mediated transcription by interfering with TFIIIB and Pol III. Herein, we found that MAF1 knockdown induced CDKN1A transcription and chromatin looping concurrently with Pol III recruitment. Simultaneous knockdown of MAF1 with Pol III or BRF1 (subunit of TFIIIB) diminished the activation and looping effect, which indicates that recruiting Pol III was required for activation of Pol II-mediated transcription and chromatin looping. Chromatin-immunoprecipitation analysis after MAF1 knockdown indicated enhanced binding of Pol III and BRF1, as well as of CFP1, p300, and PCAF, which are factors that mediate active histone marks, along with the binding of TATA binding protein (TBP) and POLR2E to the CDKN1A promoter. Simultaneous knockdown with Pol III abolished these regulatory events. Similar results were obtained for GDF15. Our results reveal a novel mechanism by which MAF1 and Pol III regulate the activity of a protein-coding gene transcribed by Pol II.

scholarly journals A TATA-Binding Protein Mutant Defective for TFIID Complex Formation In Vivo

1999 ◽  
Vol 19 (6) ◽  
pp. 3951-3957 ◽  
Author(s):  
Ryan T. Ranallo ◽  
Kevin Struhl ◽  
Laurie A. Stargell
Keyword(s):  
Binding Protein ◽  
Tata Binding Protein ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Pol Ii ◽  
Pol I ◽  
Pol Iii ◽  
Polymerase I ◽  
Tfiid Complex

ABSTRACT Using an intragenic complementation screen, we have identified a temperature-sensitive TATA-binding protein (TBP) mutant (K151L,K156Y) that is defective for interaction with certain yeast TBP-associated factors (TAFs) at the restrictive temperature. The K151L,K156Y mutant appears to be functional for RNA polymerase I (Pol I) and Pol III transcription, and it is capable of supporting Gal4-activated and Gcn4-activated transcription by Pol II. However, transcription from certain TATA-containing and TATA-less Pol II promoters is reduced at the restrictive temperature. Immunoprecipitation analysis of extracts prepared after culturing cells at the restrictive temperature for 1 h indicates that the K151L,K156Y derivative is severely compromised in its ability to interact with TAF130, TAF90, TAF68/61, and TAF25 while remaining functional for interaction with TAF60 and TAF30. Thus, a TBP mutant that is compromised in its ability to form TFIID can support the response to Gcn4 but is defective for transcription from specific promoters in vivo.

scholarly journals Regulation of rRNA Synthesis by TATA-Binding Protein-Associated Factor Mot1

2007 ◽  
Vol 27 (8) ◽  
pp. 2886-2896 ◽  
Author(s):  
Arindam Dasgupta ◽  
Rebekka O. Sprouse ◽  
Sarah French ◽  
Pavel Aprikian ◽  
Robert Hontz ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Binding Protein ◽  
Tata Binding Protein ◽  
Rrna Synthesis ◽  
Direct Role ◽  
Pol Ii ◽  
Associated Factor ◽  
Pol I

ABSTRACT Mot1 is an essential, conserved, TATA-binding protein (TBP)-associated factor in Saccharomyces cerevisiae with well-established roles in the global control of RNA polymerase II (Pol II) transcription. Previous results have suggested that Mot1 functions exclusively in Pol II transcription, but here we report a novel role for Mot1 in regulating transcription by RNA polymerase I (Pol I). In vivo, Mot1 is associated with the ribosomal DNA, and loss of Mot1 results in decreased rRNA synthesis. Consistent with a direct role for Mot1 in Pol I transcription, Mot1 also associates with the Pol I promoter in vitro in a reaction that depends on components of the Pol I general transcription machinery. Remarkably, in addition to Mot1's role in initiation, rRNA processing is delayed in mot1 cells. Taken together, these results support a model in which Mot1 affects the rate and efficiency of rRNA synthesis by both direct and indirect mechanisms, with resulting effects on transcription activation and the coupling of rRNA synthesis to processing.

scholarly journals TATA-Binding Protein Mutants That Increase Transcription from Enhancerless and Repressed Promoters In Vivo

2000 ◽  
Vol 20 (5) ◽  
pp. 1478-1488 ◽  
Author(s):  
Joseph V. Geisberg ◽  
Kevin Struhl
Keyword(s):  
Binding Protein ◽  
Tata Binding Protein ◽  
Negative Regulator ◽  
Unusual Structure ◽  
Enhancer Element ◽  
Pol Ii ◽  
Activator Proteins ◽  
Pol Iii

ABSTRACT Using a genetic screen, we isolated three TATA-binding protein (TBP) mutants that increase transcription from promoters that are repressed by the Cyc8-Tup1 or Sin3-Rpd3 corepressors or that lack an enhancer element, but not from an equivalently weak promoter with a mutated TATA element. Increased transcription is observed when the TBP mutants are expressed at low levels in the presence of wild-type TBP. These TBP mutants are unable to support cell viability, and they are toxic in strains lacking Rpd3 histone deacetylase or when expressed at higher levels. Although these mutants do not detectably bind TATA elements in vitro, genetic and chromatin immunoprecipitation experiments indicate that they act directly at promoters and do not increase transcription by titration of a negative regulatory factor(s). The TBP mutants are mildly defective for associating with promoters responding to moderate or strong activators; in addition, they are severely defective for RNA polymerase (Pol) III but not Pol I transcription. These results suggest that, with respect to Pol II transcription, the TBP mutants specifically increase expression from core promoters. Biochemical analysis indicates that the TBP mutants are unaffected for TFIID complex formation, dimerization, and interactions with either the general negative regulator NC2 or the N-terminal inhibitory domain of TAF130. We speculate that these TBP mutants have an unusual structure that allows them to preferentially access TATA elements in chromatin templates. These TBP mutants define a criterion by which promoters repressed by Cyc8-Tup1 or Sin3-Rpd3 resemble enhancerless, but not TATA-defective, promoters; hence, they support the idea that these corepressors inhibit the function of activator proteins rather than the Pol II machinery.

scholarly journals Artificially Recruited TATA-Binding Protein Fails To Remodel Chromatin and Does Not Activate Three Promoters That Require Chromatin Remodeling

2000 ◽  
Vol 20 (16) ◽  
pp. 5847-5857 ◽  
Author(s):  
Michael P. Ryan ◽  
Grace A. Stafford ◽  
Liuning Yu ◽  
Randall H. Morse
Keyword(s):  
Binding Site ◽  
Rna Polymerase Ii ◽  
Reporter Gene ◽  
Chromatin Remodeling ◽  
Chromatin Structure ◽  
Transcriptional Activation ◽  
Binding Protein ◽  
Tata Binding Protein ◽  
Activation Domains

ABSTRACT Transcriptional activators are believed to work in part by recruiting general transcription factors, such as TATA-binding protein (TBP) and the RNA polymerase II holoenzyme. Activation domains also contribute to remodeling of chromatin in vivo. To determine whether these two activities represent distinct functions of activation domains, we have examined transcriptional activation and chromatin remodeling accompanying artificial recruitment of TBP in yeast (Saccharomyces cerevisiae). We measured transcription of reporter genes with defined chromatin structure by artificial recruitment of TBP and found that a reporter gene whose TATA element was relatively accessible could be activated by artificially recruited TBP, whereas two promoters, GAL10 and CHA1, that have accessible activator binding sites, but nucleosomal TATA elements, could not. A third reporter gene containing theHIS4 promoter could be activated by GAL4-TBP only when a RAP1 binding site was present, although RAP1 alone could not activate the reporter, suggesting that RAP1 was needed to open the chromatin structure to allow activation. Consistent with this interpretation, artificially recruited TBP was unable to perturb nucleosome positioning via a nucleosomal binding site, in contrast to a true activator such as GAL4, or to perturb the TATA-containing nucleosome at theCHA1 promoter. Finally, we show that activation of theGAL10 promoter by GAL4, which requires chromatin remodeling, can occur even in swi gcn5 yeast, implying that remodeling pathways independent of GCN5, the SWI-SNF complex, and TFIID can operate during transcriptional activation in vivo.

Direct cleavage of human TATA-binding protein by poliovirus protease 3C in vivo and in vitro

1993 ◽  
Vol 13 (2) ◽  
pp. 1232-1237
Author(s):  
M E Clark ◽  
P M Lieberman ◽  
A J Berk ◽  
A Dasgupta
Keyword(s):  
Transcription Factor ◽  
Host Cell ◽  
Rna Polymerase Ii ◽  
Binding Protein ◽  
Tata Binding Protein ◽  
Significant Inhibition ◽  
Pol Ii ◽  
Poliovirus Infection

Host cell RNA polymerase II (Pol II)-mediated transcription is inhibited by poliovirus infection. This inhibition is correlated to a specific decrease in the activity of a chromatographic fraction which contains the transcription factor TFIID. To investigate the mechanism by which poliovirus infection results in a decrease of TFIID activity, we have analyzed a component of TFIID, the TATA-binding protein (TBP). Using Western immunoblot analysis, we show that TBP is cleaved in poliovirus-infected cells at the same time postinfection as when Pol II transcription is inhibited. Further, we show that one of the cleaved forms of TBP can be reproduced in vitro by incubating TBP with cloned, purified poliovirus encoded protease 3C. Protease 3C is a poliovirus-encoded protease that specifically cleaves glutamine-glycine bonds in the viral polyprotein. The cleavage of TBP by protease 3C occurs directly. Finally, incubation of an uninfected cell-derived TBP-containing fraction (TFIID) with protease 3C results in significant inhibition of Pol II-mediated transcription in vitro. These results demonstrate that a cellular transcription factor can be directly cleaved both in vitro and in vivo by a viral protease and suggest a role of the poliovirus proteinase 3C in host cell Pol II-mediated transcription shutoff.

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