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K Struhl

Publications and source records attributed to K Struhl.

At least 37 records · Page 2Linked to original sources
PubMed1999

Incorporation of Drosophila TAF110 into the yeast TFIID complex does not permit the Sp1 glutamine-rich activation domain to function in vivo.

BACKGROUND: Acidic activation domains function across eukaryotic species, and hence stimulate transcription by a conserved molecular mechanism. In contrast, glutamine-rich activation domains function in flies, mammals, and fission yeasts but not in the budding yeast Saccharomyces cerevisiae. The glutamine-rich activation domain of Sp1 interacts with TAF110, and it has been suggested that this interaction is important for transcriptional activation. S. cerevisiae does not contain a homologue of TAF110, suggesting a potential mechanism to account for the failure of glutamine-rich activation domains to stimulate transcription. RESULTS: Here, we have artificially recruited Drosophila TAF110 into the yeast TFIID complex by fusing it to yeast TBP. The resulting TFIID complex supports normal cell growth, but it is unable to mediate Sp1-dependent activation. CONCLUSIONS: Thus, the interaction of glutamine-rich activation domains with TAF110 is insufficient for transcriptional activation in vivo, indicating that other targets within the PolII machinery are necessary.

Animals↗
PubMed1999

Transcriptional activation in yeast cells lacking transcription factor IIA.

The general transcription factor IIA (TFIIA) forms a complex with TFIID at the TATA promoter element, and it inhibits the function of several negative regulators of the TATA-binding protein (TBP) subunit of TFIID. Biochemical experiments suggest that TFIIA is important in the response to transcriptional activators because activation domains can interact with TFIIA, increase recruitment of TFIID and TFIIA to the promoter, and promote isomerization of the TFIID-TFIIA-TATA complex. Here, we describe a double-shut-off approach to deplete yeast cells of Toa1, the large subunit of TFIIA, to <1% of the wild-type level. Interestingly, such TFIIA-depleted cells are essentially unaffected for activation by heat shock factor, Ace1, and Gal4-VP16. However, depletion of TFIIA causes a general two- to threefold decrease of transcription from most yeast promoters and a specific cell-cycle arrest at the G2-M boundary. These results indicate that transcriptional activation in vivo can occur in the absence of TFIIA.

Cell Cycle↗
PubMed1999

A TATA-binding protein mutant defective for TFIID complex formation in vivo.

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.

Amitrole↗
PubMed1998

Association of distinct yeast Not2 functional domains with components of Gcn5 histone acetylase and Ccr4 transcriptional regulatory complexes.

The NOT genes were originally identified in a yeast genetic screen that selected mutations resulting in increased utilization of a non-consensus TC TATA element of the HIS3 promoter. Here, we present evidence that the N-terminus of Not2 interacts with components of the Ada/Gcn5 histone acetyltransferase complex. Loss of this interaction either through abrogation of Not2 N-terminal function or deletion of ada2 or gcn5 results in derepression of the HIS3 TC element. This suggests that association of Not2 with the Ada/Gcn5 histone acetyltransferase complex is involved in regulation of the HIS3 promoter. Association between the Not and CCR4 transcriptional regulatory complexes has also been observed recently. Our phenotypic analyses suggest that these CCR4-related Not2 functions are mediated by a functionally independent domain of Not2 that includes the highly conserved C-terminus. Chimeric proteins containing the yeast Not2 N-terminus fused to the human C-terminus function in yeast, suggesting that the Not2 C-terminus represents a distinct modular domain whose function is conserved between higher and lower eukaryotes.

Acetyltransferases↗
PubMed1998

Histone deacetylase activity of Rpd3 is important for transcriptional repression in vivo.

Eukaryotic organisms from yeast to human contain a multiprotein complex that includes Rpd3 histone deacetylase and Sin3 corepressor. The Sin3-Rpd3 complex, when recruited to promoters by specific DNA-binding proteins, can direct transcriptional repression of specific classes of target genes. It has been proposed that the histone deacetylase activity of Rpd3 is important for repression, but direct evidence is lacking. Here, we describe four Rpd3 derivatives with mutations in evolutionarily invariant histidine residues in a putative deacetylation motif. These Rpd3 mutants lack detectable histone deacetylase activity in vitro, but interact normally with Sin3 in vivo. In yeast cells, these catalytically inactive mutants are defective for transcriptional repression. They retain some residual Rpd3 function in vivo, however, suggesting that repression by the Sin3-Rpd3 complex may not be attributable exclusively to its intrinsic histone deacetylase activity. Finally, we show that a human Rpd3 homolog can interact with yeast Sin3 and repress transcription when artificially recruited to a promoter. These results suggest that the histone deacetylase activity of Rpd3 is important, but perhaps not absolutely required, for transcriptional repression in vivo.

Acetylation↗
PubMed1998

Activator-mediated recruitment of the RNA polymerase II machinery is the predominant mechanism for transcriptional activation in yeast.

Eukaryotic transcriptional activators bind to enhancer elements and stimulate the RNA polymerase II (pol II) machinery via functionally autonomous activation domains. In yeast cells, the normal requirement for an activation domain can be bypassed by artificially connecting an enhancer-bound protein to a component of the pol II machinery. This observation suggests, but does not necessarily indicate, that the physiological role of activation domains is to recruit the pol II apparatus to promoters. Here, we show that transcriptional stimulation does not occur when the activation domain is physically disconnected from the enhancer-bound protein and transferred to components of the pol II machinery. The observation that autonomous activation domains are functional when connected to enhancer-bound proteins but not to components of the pol II machinery strongly argues that recruitment is the predominant mechanism for transcriptional activation in yeast.

Bacterial Proteins↗
PubMed1998

The histone H3-like TAF is broadly required for transcription in yeast.

In yeast cells, independent depletion of TAFs (130, 67, 40, and 19) found specifically in TFIID results in selective effects on transcription, including a common effect on his3 core promoter function. In contrast, depletion of TAF17, which is also present in the SAGA histone acetylase complex, causes a decrease in transcription of most genes. However, TAF17-depleted cells maintain Ace1-dependent activation, and they induce de novo activation by heat shock factor in a manner predominantly associated with the activator, not the core promoter. Thus, TAF17 is broadly, but not universally, required for transcription in yeast, TAF17 depletion and TAF130 depletion each disrupt TFIID integrity yet cause different transcriptional consequences, suggesting that the widespread influence of TAF17 might not be due solely to its function in TFIID.

Blotting, Western↗
PubMed1998

The mating-type proteins of fission yeast induce meiosis by directly activating mei3 transcription.

Cell type control of meiotic gene regulation in the budding yeast Saccharomyces cerevisiae is mediated by a cascade of transcriptional repressors, a1-alpha2 and Rme1. Here, we investigate the analogous regulatory pathway in the fission yeast Schizosaccharomyces pombe by analyzing the promoter of mei3, the single gene whose expression is sufficient to trigger meiosis. The mei3 promoter does not appear to contain a negative regulatory element that represses transcription in haploid cells. Instead, correct regulation of mei3 transcription depends on a complex promoter that contains at least five positive elements upstream of the TATA sequence. These elements synergistically activate mei3 transcription, thereby constituting an on-off switch for the meiosis pathway. Element C is a large region containing multiple sequences that resemble binding sites for Mc, an HMG domain protein encoded by the mating-type locus. The function of element C is extremely sensitive to spacing changes but not to linker-scanning mutations, suggesting the possibility that Mc functions as an architectural transcription factor. Altered-specificity experiments indicate that element D interacts with Pm, a homeodomain protein encoded by the mating-type locus. This indicates that Pm functions as a direct activator of the meiosis pathway, whereas the homologous mating-type protein in S. cerevisiae (alpha2) functions as a repressor. Thus, despite the strong similarities between the mating-type loci of S. cerevisiae and S. pombe, the regulatory logic that governs the tight control of the key meiosis-inducing genes in these organisms is completely different.

Base Sequence↗
PubMed1998

Targeted recruitment of the Sin3-Rpd3 histone deacetylase complex generates a highly localized domain of repressed chromatin in vivo.

Eukaryotic organisms contain a multiprotein complex that includes Rpd3 histone deacetylase and the Sin3 corepressor. The Sin3-Rpd3 complex is recruited to promoters by specific DNA-binding proteins, whereupon it represses transcription. By directly analyzing the chromatin structure of a repressed promoter in yeast cells, we demonstrate that transcriptional repression is associated with localized histone deacetylation. Specifically, we observe decreased acetylation of histones H3 and H4 (preferentially lysines 5 and 12) that depends on the DNA-binding repressor (Ume6), Sin3, and Rpd3. Mapping experiments indicate that the domain of histone deacetylation is highly localized, occurring over a range of one to two nucleosomes. Taken together with previous observations, these results define a novel mechanism of transcriptional repression which involves targeted recruitment of a histone-modifying activity and localized perturbation of chromatin structure.

Chromatin↗
PubMed1997

Repression by Ume6 involves recruitment of a complex containing Sin3 corepressor and Rpd3 histone deacetylase to target promoters.

Sin3 and Rpd3 negatively regulate a diverse set of yeast genes. A mouse Sin3-related protein is a transcriptional corepressor, and a human Rpd3 homolog is a histone deacetylase. Here, we show that Sin3 and Rpd3 are specifically required for transcriptional repression by Ume6, a DNA-binding protein that regulates genes involved in meiosis. A short region of Ume6 is sufficient to repress transcription, and this repression domain mediates a two-hybrid and physical interaction with Sin3. Coimmunoprecipitation and two-hybrid experiments indicate that Sin3 and Rpd3 are associated in a complex distinct from TFIID and Pol II holoenzyme. Rpd3 is specifically required for repression by Sin3, and artificial recruitment of Rpd3 results in repression. These results suggest that repression by Ume6 involves recruitment of a Sin3-Rpd3 complex and targeted histone deacetylation.

DNA-Binding Proteins↗
PubMed1997

Characterisation of 3' end formation of the yeast HIS3 mRNA.

The nucleotide (nt) sequence of the 3' end of the yeast HIS3 mRNA was determined by PCR amplification of the 3' end. Analysis of 28 individual clones revealed that at least 13 distinct polyadenylation sites are present. The sites of polyadenylation are extremely heterogeneous and do not show any obvious similarity other than that they occur after pyrimidine residues in most cases. Most mutants carrying internal deletions of the 3' untranslated region (3' UTR) did not abolish 3' end formation and showed polyadenylation at normal sites. Deletion of a 90-nt region that contains an A+T-rich sequence close to the 3' end of the HIS3 coding sequence and a subset of processing sites resulted in a drastic reduction in the levels of full-length HIS3 mRNA and concomitant transcription past the normal HIS3 3' end. The 90-nt region appears to be sufficient to direct the formation of at least a subset of the HIS3 3' ends since mutants that carry deletions of flanking regions of this sequence show detectable levels of HIS3 mRNA. Spacing between the upstream A-T sequence and the site of processing is variable. In the light of the extreme heterogeneity of the sites, a possible mechanism for 3' processing is discussed.

Base Sequence↗
PubMed1997

Selective roles for TATA-binding-protein-associated factors in vivo.

Transcription factor TFIID, a central component of the eukaryotic RNA polymerase II transcription machinery, is a multiprotein complex containing the TATA-binding protein (TBP) and TBP-associated factors (TAFs). In vitro, TAFs are required for the response to activator proteins, but they are dispensible for basal transcription. However, recent work in yeast cells indicates that TAFs are not generally required for transcriptional activation, but rather have selective effects on gene expression. Molecular mechanisms for these observations are considered.

DNA-Binding Proteins↗
PubMed1997

Transcriptional activation by TFIIB mutants that are severely impaired in interaction with promoter DNA and acidic activation domains.

Biochemical experiments indicate that the general transcription factor IIB (TFIIB) can interact directly with acidic activation domains and that activators can stimulate transcription by increasing recruitment of TFIIB to promoters. For promoters at which recruitment of TFIIB to promoters is limiting in vivo, one would predict that transcriptional activity should be particularly sensitive to TFIIB mutations that decrease the association of TFIIB with promoter DNA and/or with activation domains; i.e., such TFIIB mutations should exacerbate a limiting step that occurs in wild-type cells. Here, we describe mutations on the DNA-binding surface of TFIIB that severely affect both TATA-binding protein (TBP)-TFIIB-TATA complex formation and interaction with the VP16 activation domain in vitro. These TFIIB mutations affect the stability of the TBP-TFIIB-TATA complex in vivo because they are synthetically lethal in combination with TBP mutants impaired for TFIIB binding. Interestingly, these TFIIB derivatives support viability, and they efficiently respond to Gal4-VP16 and natural acidic activators in different promoter contexts. These results suggest that in vivo, recruitment of TFIIB is not generally a limiting step for acidic activators. However, one TFIIB derivative shows reduced transcription of GAL4, suggesting that TFIIB may be limiting at a subset of promoters in vivo.

Amino Acid Sequence↗
PubMed1997

Yap, a novel family of eight bZIP proteins in Saccharomyces cerevisiae with distinct biological functions.

Saccharomyces cerevisiae contains eight members of a novel and fungus-specific family of bZIP proteins that is defined by four atypical residues on the DNA-binding surface. Two of these proteins, Yap1 and Yap2, are transcriptional activators involved in pleiotropic drug resistance. Although initially described as AP-1 factors, at least four Yap proteins bind most efficiently to TTACTAA, a sequence that differs at position +/-2 from the optimal AP-1 site (TGACTCA); further, a Yap-like derivative of the AP-1 factor Gcn4 (A239Q S242F) binds efficiently to the Yap recognition sequence. Molecular modeling suggests that the Yap-specific residues make novel contacts and cause physical constraints at the +/-2 position that may account for the distinct DNA-binding specificities of Yap and AP-1 proteins. To various extents, Yap1, Yap2, Yap3, and Yap5 activate transcription from a promoter containing a Yap recognition site. Yap-dependent transcription is abolished in strains containing high levels of protein kinase A; in contrast, Gcn4 transcriptional activity is stimulated by protein kinase A. Interestingly, Yap1 transcriptional activity is stimulated by hydrogen peroxide, whereas Yap2 activity is stimulated by aminotriazole and cadmium. In addition, unlike other yap mutations tested, yap4 (cin5) mutations affect chromosome stability, and they suppress the cold-sensitive phenotype of yap1 mutant strains. Thus, members of the Yap family carry out overlapping but distinct biological functions.

Amino Acid Sequence↗
PubMed1997

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

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.

Alanine↗