scholarly journals Analysis of TFIIA Function In Vivo: Evidence for a Role in TATA-Binding Protein Recruitment and Gene-Specific Activation

1999 ◽  
Vol 19 (12) ◽  
pp. 8673-8685 ◽  
Author(s):  
Qing Liu ◽  
Scott E. Gabriel ◽  
Kelli L. Roinick ◽  
Robert D. Ward ◽  
Karen M. Arndt
Keyword(s):  
Dna Binding ◽  
Binding Protein ◽  
Gene Activation ◽  
Tata Binding Protein ◽  
New Class ◽  
Activation Of Transcription ◽  
Protein Recruitment ◽  
Dna Complex

ABSTRACT Activation of transcription can occur by the facilitated recruitment of TFIID to promoters by gene-specific activators. To investigate the role of TFIIA in TFIID recruitment in vivo, we exploited a class of yeast TATA-binding protein (TBP) mutants that is activation and DNA binding defective. We found that co-overexpression of TOA1 and TOA2, the genes that encode yeast TFIIA, overcomes the activation defects caused by the TBP mutants. Using a genetic screen, we isolated a new class of TFIIA mutants and identified three regions on TFIIA that are likely to be involved in TBP recruitment or stabilization of the TBP-TATA complex in vivo. Amino acid replacements in only one of these regions enhance TFIIA-TBP-DNA complex formation in vitro, suggesting that the other regions are involved in regulatory interactions. To determine the relative importance of TFIIA in the regulation of different genes, we constructed yeast strains to conditionally deplete TFIIA levels prior to gene activation. While the activation of certain genes, such asINO1, was dramatically impaired by TFIIA depletion, activation of other genes, such as CUP1, was unaffected. These data suggest that TFIIA facilitates DNA binding by TBP in vivo, that TFIIA may be regulated by factors that target distinct regions of the protein, and that promoters vary significantly in the degree to which they require TFIIA for activation.

scholarly journals A Human TATA Binding Protein-Related Protein with Altered DNA Binding Specificity Inhibits Transcription from Multiple Promoters and Activators

1999 ◽  
Vol 19 (11) ◽  
pp. 7610-7620 ◽  
Author(s):  
Paul A. Moore ◽  
Josef Ozer ◽  
Moreh Salunek ◽  
Gwenael Jan ◽  
Dennis Zerby ◽  
...  
Keyword(s):  
Dna Binding ◽  
Transcriptional Repression ◽  
Transcription Initiation ◽  
Binding Protein ◽  
Tata Binding Protein ◽  
Related Protein ◽  
Multiple Promoters ◽  
Binding Surface

ABSTRACT The TATA binding protein (TBP) plays a central role in eukaryotic and archael transcription initiation. We describe the isolation of a novel 23-kDa human protein that displays 41% identity to TBP and is expressed in most human tissue. Recombinant TBP-related protein (TRP) displayed barely detectable binding to consensus TATA box sequences but bound with slightly higher affinities to nonconsensus TATA sequences. TRP did not substitute for TBP in transcription reactions in vitro. However, addition of TRP potently inhibited basal and activated transcription from multiple promoters in vitro and in vivo. General transcription factors TFIIA and TFIIB bound glutathioneS-transferase–TRP in solution but failed to stimulate TRP binding to DNA. Preincubation of TRP with TFIIA inhibited TBP-TFIIA-DNA complex formation and addition of TFIIA overcame TRP-mediated transcription repression. TRP transcriptional repression activity was specifically reduced by mutations in TRP that disrupt the TFIIA binding surface but not by mutations that disrupt the TFIIB or DNA binding surface of TRP. These results suggest that TFIIA is a primary target of TRP transcription inhibition and that TRP may modulate transcription by a novel mechanism involving the partial mimicry of TBP functions.

scholarly journals Multiple Functions of the Nonconserved N-Terminal Domain of Yeast TATA-Binding Protein

Genetics ◽  
2001 ◽  
Vol 158 (1) ◽  
pp. 87-93
Author(s):  
Mark Lee ◽  
Kevin Struhl
Keyword(s):  
Dna Binding ◽  
Binding Protein ◽  
Protein S ◽  
Tata Binding Protein ◽  
Terminal Region ◽  
Core Domain ◽  
Terminal Domain ◽  
Two Factors

Abstract The TATA-binding protein (TBP) is composed of a highly conserved core domain sufficient for TATA-element binding and preinitiation complex formation as well as a highly divergent N-terminal region that is dispensable for yeast cell viability. In vitro, removal of the N-terminal region domain enhances TBP-TATA association and TBP dimerization. Here, we examine the effects of truncation of the N-terminal region in the context of yeast TBP mutants with specific defects in DNA binding and in interactions with various proteins. For a subset of mutations that disrupt DNA binding and the response to transcriptional activators, removal of the N-terminal domain rescues their transcriptional defects. By contrast, deletion of the N-terminal region is lethal in combination with mutations on a limited surface of TBP. Although this surface is important for interactions with TFIIA and Brf1, TBP interactions with these two factors do not appear to be responsible for this dependence on the N-terminal region. Our results suggest that the N-terminal region of TBP has at least two distinct functions in vivo. It inhibits the interaction of TBP with TATA elements, and it acts positively in combination with a specific region of the TBP core domain that presumably interacts with another protein(s).

scholarly journals Role for Nhp6, Gcn5, and the Swi/Snf Complex in Stimulating Formation of the TATA-Binding Protein-TFIIA-DNA Complex

2004 ◽  
Vol 24 (18) ◽  
pp. 8312-8321 ◽  
Author(s):  
Debabrata Biswas ◽  
Anthony N. Imbalzano ◽  
Peter Eriksson ◽  
Yaxin Yu ◽  
David J. Stillman
Keyword(s):  
Binding Protein ◽  
Tata Binding Protein ◽  
Growth Defect ◽  
Saga Complex ◽  
Transcriptional Initiation ◽  
Genes Encoding ◽  
Promoter Dna ◽  
Dna Complex

ABSTRACT The TATA-binding protein (TBP), TFIIA, and TFIIB interact with promoter DNA to form a complex required for transcriptional initiation, and many transcriptional regulators function by either stimulating or inhibiting formation of this complex. We have recently identified TBP mutants that are viable in wild-type cells but lethal in the absence of the Nhp6 architectural transcription factor. Here we show that many of these TBP mutants were also lethal in strains with disruptions of either GCN5, encoding the histone acetyltransferase in the SAGA complex, or SWI2, encoding the catalytic subunit of the Swi/Snf chromatin remodeling complex. These synthetic lethalities could be suppressed by overexpression of TOA1 and TOA2, the genes encoding TFIIA. We also used TFIIA mutants that eliminated in vitro interactions with TBP. These viable TFIIA mutants were lethal in strains lacking Gcn5, Swi2, or Nhp6. These lethalities could be suppressed by overexpression of TBP or Nhp6, suggesting that these coactivators stimulate formation of the TBP-TFIIA-DNA complex. In vitro studies have previously shown that TBP binds very poorly to a TATA sequence within a nucleosome but that Swi/Snf stimulates binding of TBP and TFIIA. In vitro binding experiments presented here show that histone acetylation facilitates TBP binding to a nucleosomal binding site and that Nhp6 stimulates formation of a TBP-TFIIA-DNA complex. Consistent with the idea that Nhp6, Gcn5, and Swi/Snf have overlapping functions in vivo, nhp6a nhp6b gcn5 mutants had a severe growth defect, and mutations in both nhp6a nhp6b swi2 and gcn5 swi2 strains were lethal.

scholarly journals Molecular analysis of the SNF2/SWI2 protein family member MOT1, an ATP-driven enzyme that dissociates TATA-binding protein from DNA.

1997 ◽  
Vol 17 (8) ◽  
pp. 4842-4851 ◽  
Author(s):  
D T Auble ◽  
D Wang ◽  
K W Post ◽  
S Hahn
Keyword(s):  
Conformational Changes ◽  
Binding Protein ◽  
Tata Binding Protein ◽  
Protein Family ◽  
Power Stroke ◽  
Chimeric Proteins ◽  
Protein Dna Interactions ◽  
Dna Complex

MOT1 is an essential Saccharomyces cerevisiae protein and a member of the SNF2/SWI2 family of ATPases. MOT1 functions by removing TATA-binding protein (TBP) from DNA, and as a consequence, MOT1 can regulate transcription both in vitro and in vivo. Here we describe the in vivo and in vitro activities of MOT1 deletion and substitution mutants. The results indicate that MOT1 is targeted to TBP both in vitro and in vivo via amino acids in its nonconserved N terminus. The conserved C-terminal ATPase of MOT1 appears to contribute to TBP-DNA complex recognition in the absence of ATP, but it appears to function primarily during the actual ATP-dependent dissociation reaction. Chimeric proteins in which homologous portions of SNF2/SWI2 have been substituted for the MOT1 ATPase can bind to TBP-DNA complexes but fail to dissociate these complexes in the presence of ATP, suggesting that the specificity of action of MOT1 is also conferred by the C-terminal ATPase. ATPase assays demonstrate that the MOT1 ATPase is activated by TBP. Thus, MOT1 undergoes at least two conformational changes: (i) an allosteric effect of TBP that mediates the activation of the MOT1 ATPase and (ii) an ATP-driven "power stroke" that causes TBP-DNA complex dissociation. These results provide a general framework for understanding how members of the SNF2/SWI2 protein family use ATP to modulate protein-DNA interactions to regulate many diverse processes in cells.

scholarly journals A severely defective TATA-binding protein-TFIIB interaction does not preclude transcriptional activation in vivo.

1997 ◽  
Vol 17 (3) ◽  
pp. 1336-1345 ◽  
Author(s):  
M Lee ◽  
K Struhl
Keyword(s):  
Dna Binding ◽  
Complex Formation ◽  
Transcriptional Activation ◽  
Binding Protein ◽  
Tata Binding Protein ◽  
Yeast Cells ◽  
Synthetic Lethal ◽  
Binding Surface

In yeast cells, mutations in the TATA-binding protein (TBP) that disrupt the interaction with the TATA element or with TFIIA can selectively impair the response to acidic activator proteins. We analyzed the transcriptional properties of TBP derivatives in which residues that directly interact with TFIIB were replaced by alanines. Surprisingly, a derivative with a 50-fold defect in TBP-TFIIB-TATA complex formation in vitro (E188A) supports viability and responds efficiently to activators in vivo. The E186A derivative, which displays a 100-fold defect in TBP-TFIIB-TATA complex formation, does not support viability, yet it does respond to activators. Conversely, the L189A mutation, which has the mildest effect on the interaction with TFIIB (10-fold), can abolish transcriptional activation and cell viability when combined with mutations on the DNA-binding surface. This "synthetic lethal" effect is not observed with E188A, suggesting that the previously described role of L189 in transcriptional activation may be related to its location on the DNA-binding surface and not to its interaction with TFIIB. Finally, when using TBP mutants defective on multiple interaction surfaces, we observed synthetic lethal effects between mutations on the TFIIA and TFIIB interfaces but found that mutations implicated in association with polymerase II and TFIIF did not have significant effects in vivo. Taken together, these results argue that, unlike the TBP-TATA and TBP-TFIIA interactions, the TBP-TFIIB interaction is not generally limiting for transcriptional activation in vivo.

Novel pathway for thyroid hormone receptor action through interaction with jun and fos oncogene activities

1991 ◽  
Vol 11 (12) ◽  
pp. 6016-6025
Author(s):  
X K Zhang ◽  
K N Wills ◽  
M Husmann ◽  
T Hermann ◽  
M Pfahl
Keyword(s):  
Signal Transduction ◽  
Dna Binding ◽  
Thyroid Hormone Receptor ◽  
Gene Activation ◽  
Signal Transduction Pathway ◽  
Large Family ◽  
Transduction Pathway ◽  
Regulatory Pathway

Many essential biological pathways, including cell growth, development, and metabolism, are regulated by thyroid hormones (THs). TH action is mediated by intracellular receptors that belong to a large family of ligand-dependent transcription factors, including the steroid hormone and retinoic acid receptors. So far it has been assumed that TH receptors (TRs) regulate gene transcription only through the classical protein-DNA interaction mechanism. Here we provide evidence for a regulatory pathway that allows cross-talk between TRs and the signal transduction pathway used by many growth factors, oncogenes, and tumor promoters. In transient transfection studies, we observed that the oncogenes c-jun and c-fos inhibit TR activities, while TRs inhibit induction of the c-fos promoter and repress AP-1 site-dependent gene activation. A truncated TR that lacks only 17 amino acids from the carboxy terminus can no longer antagonize AP-1 activity. The cross-regulation between TRs and the signal transduction pathway appears to be based on the ability of TRs to inhibit DNA binding of the transcription factor AP-1 in the presence of THs. The constituents of AP-1, c-Jun, and c-Fos, vice versa, can inhibit TR-induced gene activation in vivo, and c-Jun inhibits TR DNA binding in vitro. This novel regulatory pathway is likely to play a major role in growth control and differentiation by THs.

Mechanism of initiator-mediated transcription: evidence for a functional interaction between the TATA-binding protein and DNA in the absence of a specific recognition sequence

1993 ◽  
Vol 13 (7) ◽  
pp. 3841-3849
Author(s):  
B Zenzie-Gregory ◽  
A Khachi ◽  
I P Garraway ◽  
S T Smale
Keyword(s):  
Transcription Initiation ◽  
Binding Protein ◽  
Tata Binding Protein ◽  
Upstream Region ◽  
Promoter Strength ◽  
Recognition Sequence ◽  
Specific Recognition ◽  
Rate Limiting

Promoters containing Sp1 binding sites and an initiator element but lacking a TATA box direct high levels of accurate transcription initiation by using a mechanism that requires the TATA-binding protein (TBP). We have begun to address the role of TBP during transcription from Sp1-initiator promoters by varying the nucleotide sequence between -14 and -33 relative to the start site. With each of several promoters containing different upstream sequences, we detected accurate transcription both in vitro and in vivo, but the promoter strengths varied widely, particularly with the in vitro assay. The variable promoter activities correlated with, but were not proportional to, the abilities of the upstream sequences to function as TATA boxes, as assessed by multiple criteria. These results confirm that accurate transcription can proceed in the presence of an initiator, regardless of the sequence present in the -30 region. However, the results reveal a role for this upstream region, most consistent with a model in which initiator-mediated transcription requires binding of TBP to the upstream DNA in the absence of a specific recognition sequence. Moreover, in vivo it appears that the promoter strength is modulated less severely by altering the -30 sequence, consistent with a previous suggestion that TBP is not rate limiting in vivo for TATA-less promoters. Taken together, these results suggest that variations in the structure of a core promoter might alter the rate-limiting step for transcription initiation and thereby alter the potential modes of transcriptional regulation, without severely changing the pathway used to assemble a functional preinitiation complex.

scholarly journals Functional Organization of the Yeast SAGA Complex: Distinct Components Involved in Structural Integrity, Nucleosome Acetylation, and TATA-Binding Protein Interaction

1999 ◽  
Vol 19 (1) ◽  
pp. 86-98 ◽  
Author(s):  
David E. Sterner ◽  
Patrick A. Grant ◽  
Shannon M. Roberts ◽  
Laura J. Duggan ◽  
Rimma Belotserkovskaya ◽  
...  
Keyword(s):  
Histone Acetylation ◽  
Structural Integrity ◽  
Functional Organization ◽  
Binding Protein ◽  
Tata Binding Protein ◽  
Saga Complex ◽  
Specific Domain ◽  
Nucleosomal Histone

ABSTRACT SAGA, a recently described protein complex in Saccharomyces cerevisiae, is important for transcription in vivo and possesses histone acetylation function. Here we report both biochemical and genetic analyses of members of three classes of transcription regulatory factors contained within the SAGA complex. We demonstrate a correlation between the phenotypic severity of SAGA mutants and SAGA structural integrity. Specifically, null mutations in the Gcn5/Ada2/Ada3 or Spt3/Spt8 classes cause moderate phenotypes and subtle structural alterations, while mutations in a third subgroup, Spt7/Spt20, as well as Ada1, disrupt the complex and cause severe phenotypes. Interestingly, double mutants (gcn5Δ spt3Δand gcn5Δ spt8Δ) causing loss of a member of each of the moderate classes have severe phenotypes, similar tospt7Δ, spt20Δ, or ada1Δmutants. In addition, we have investigated biochemical functions suggested by the moderate phenotypic classes and find that first, normal nucleosomal acetylation by SAGA requires a specific domain of Gcn5, termed the bromodomain. Deletion of this domain also causes specific transcriptional defects at the HIS3 promoter in vivo. Second, SAGA interacts with TBP, the TATA-binding protein, and this interaction requires Spt8 in vitro. Overall, our data demonstrate that SAGA harbors multiple, distinct transcription-related functions, including direct TBP interaction and nucleosomal histone acetylation. Loss of either of these causes slight impairment in vivo, but loss of both is highly detrimental to growth and transcription.

scholarly journals The HOX Homeodomain Proteins Block CBP Histone Acetyltransferase Activity

2001 ◽  
Vol 21 (21) ◽  
pp. 7509-7522 ◽  
Author(s):  
Wei-fang Shen ◽  
Keerthi Krishnan ◽  
H. J. Lawrence ◽  
Corey Largman
Keyword(s):  
Dna Binding ◽  
Protein Function ◽  
Histone Acetyltransferase ◽  
Gene Activation ◽  
Homeodomain Proteins ◽  
Hox Proteins ◽  
Reporter Assays ◽  
Hox Protein

ABSTRACT Despite the identification of PBC proteins as cofactors that provide DNA affinity and binding specificity for the HOX homeodomain proteins, HOX proteins do not demonstrate robust activity in transient-transcription assays and few authentic downstream targets have been identified for these putative transcription factors. During a search for additional cofactors, we established that each of the 14 HOX proteins tested, from 11 separate paralog groups, binds to CBP or p300. All six isolated homeodomain fragments tested bind to CBP, suggesting that the homeodomain is a common site of interaction. Surprisingly, CBP-p300 does not form DNA binding complexes with the HOX proteins but instead prevents their binding to DNA. The HOX proteins are not substrates for CBP histone acetyltransferase (HAT) but instead inhibit the activity of CBP in both in vitro and in vivo systems. These mutually inhibitory interactions are reflected by the inability of CBP to potentiate the low levels of gene activation induced by HOX proteins in a range of reporter assays. We propose two models for HOX protein function: (i) HOX proteins may function without CBP HAT to regulate transcription as cooperative DNA binding molecules with PBX, MEIS, or other cofactors, and (ii) the HOX proteins may inhibit CBP HAT activity and thus function as repressors of gene transcription.

scholarly journals The DNA-binding protein HTa from Thermoplasma acidophilum is an archaeal histone analog

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Antoine Hocher ◽  
Maria Rojec ◽  
Jacob B Swadling ◽  
Alexander Esin ◽  
Tobias Warnecke
Keyword(s):  
Dna Binding ◽  
Binding Protein ◽  
Dna Binding Protein ◽  
Coli Strain ◽  
Micrococcal Nuclease ◽  
Thermoplasma Acidophilum ◽  
Chromatin Architecture ◽  
Archaeal Histone

Histones are a principal constituent of chromatin in eukaryotes and fundamental to our understanding of eukaryotic gene regulation. In archaea, histones are widespread but not universal: several lineages have lost histone genes. What prompted or facilitated these losses and how archaea without histones organize their chromatin remains largely unknown. Here, we elucidate primary chromatin architecture in an archaeon without histones, Thermoplasma acidophilum, which harbors a HU family protein (HTa) that protects part of the genome from micrococcal nuclease digestion. Charting HTa-based chromatin architecture in vitro, in vivo and in an HTa-expressing E. coli strain, we present evidence that HTa is an archaeal histone analog. HTa preferentially binds to GC-rich sequences, exhibits invariant positioning throughout the growth cycle, and shows archaeal histone-like oligomerization behavior. Our results suggest that HTa, a DNA-binding protein of bacterial origin, has converged onto an architectural role filled by histones in other archaea.

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