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K Struhl

Publications and source records attributed to K Struhl.

At least 55 records · Page 3Linked to original sources
PubMed1996

The histone deacetylase RPD3 counteracts genomic silencing in Drosophila and yeast.

Both position-effect variegation (PEV) in Drosophila and telomeric position-effect in yeast (TPE) result from the mosaic inactivation of genes relocated next to a block of centromeric heterochromatin or next to telomeres. In many aspects, these phenomena are analogous to other epigenetic silencing mechanisms, such as the control of homeotic gene clusters, X-chromosome inactivation and imprinting in mammals, and mating-type control in yeast. Dominant mutations that suppress or enhance PEV are thought to encode either chromatin proteins or factors that directly affect chromatin structure. We have identified an insertional mutation in Drosophila that enhances PEV and reduces transcription of the gene in the eye-antenna imaginal disc. The gene corresponds to that encoding the transcriptional regulator RPD3 in yeast, and to a human histone deacetylase. In yeast, RRD3-deletion strains show enhanced TPE, suggesting a conserved role of the histone deacetylase RPD3 in counteracting genomic silencing. This function of RPD3, which is in contrast to the general correlation between histone acetylation and increased transcription, might be due to a specialized chromatin structure at silenced loci.

Amino Acid Sequence↗
PubMed1996

Yeast homologues of higher eukaryotic TFIID subunits.

In eukaryotic cells the TATA-binding protein (TBP) associates with other proteins known as TBP-associated factors (TAFs) to form multisubunit transcription factors important for gene expression by all three nuclear RNA polymerases. Computer searching of the complete Saccharomyces cerevisiae genome revealed five previously unidentified yeast genes with significant sequence similarity to known human and Drosophila RNA polymerase II TAFs. Each of these genes is essential for viability. A sixth essential gene (FUN81) has previously been noted to be similar to human TAFII18. Coimmunoprecipitation experiments show that all six proteins are associated with TBP, demonstrating that they are true TAFs. Furthermore, these proteins are present in complexes containing the TAFII130 subunit, indicating that they are components of TFIID. Based on their predicted molecular weights, these genes have been designated TAF67, TAF61(68), TAF40, TAF23(25), TAF19(FUN81), and TAF17. Yeast TAF61 is significantly larger than its higher eukaryotic homologues, and deletion analysis demonstrates that the evolutionarily conserved, histone-like domain is sufficient and necessary to support viability.

Amino Acid Sequence↗
PubMed1996

TBP-associated factors are not generally required for transcriptional activation in yeast.

The transcription factor TFIID, a central component of the eukaryotic RNA polymerase II (Pol II) transcription apparatus, comprises the TATA-binding protein (TBP) and approximately ten TBP-associated factors (TAFs). Although the essential role of TBP in all eukaryotic transcription has been extensively analysed in vivo and in vitro, the function of the TAFs is less clear. In vitro, TAFs are dispensable for basal transcription but are required for the response to activators. In addition, specific TAFs may act as molecular bridges between particular activators and the general transcription machinery. In vivo, TAFS are required for yeast and mammalian cell growth, but little is known about their specific transcriptional functions. Using conditional alleles created by a new double-shutoff method, we show here that TAF depletion in yeast cells can reduce transcription from some promoters lacking conventional TATA elements. However, TAF depletion has surprisingly little effect on transcriptional enhancement by several activators, indicating that TAFs are not generally required for transcriptional activation in yeast.

Alleles↗
PubMed1996

Absolute mRNA levels and transcriptional initiation rates in Saccharomyces cerevisiae.

We quantitate the absolute levels of individual mRNAs per yeast cell by hybridizing total yeast RNA with an excess of gene-specific 32P-oligonucleotides, and digesting the resulting RNA-DNA hybrids with S1 nuclease. By comparing the his3 hybridization signal from a known amount of yeast cells to the signal generated by a known amount of his3 RNA synthesized in vitro, we determine that yeast strain KY114 growing in yeast extract/peptone/glucose medium at 30 degrees C contains seven molecules of his3 mRNA per cell. Using a galactose shut-off procedure, we determined that the half-life of his3 mRNA is approximately 11 min under these conditions. From these observations, we calculate that one his3 mRNA molecule is synthesized every 140 s. Analysis of other his3 promoter derivatives suggests that the maximal transcriptional initiation rate in yeast cells is one mRNA molecule every 6-8 s. Using his3 as an internal standard, the number of mRNA molecules per cell have been determined for ded1, trp3, rps4, and gall under a variety of growth conditions. From these results, the absolute mRNA level of any yeast gene can be determined in a single hybridization experiment. Moreover, the rate of transcriptional initiation can be determined for mRNAs whose decay rates are known.

Base Sequence↗
PubMed1996

Mechanisms of transcriptional activation in vivo: two steps forward.

Transcriptional activation involves the regulated assembly of multiprotein complexes on promoter DNA in the context of the repressive effects of chromatin. How do activators orchestrate this complicated phenomenon in vivo? Recent genetic and biochemical advancements suggest that activator-dependent formation of the transcription machinery on the promoter involves at least two steps. First, the activator facilitates the recruitment of TFIID to the TATA element of the promoter. TFIID binding is then followed by the recruitment of the remainder of the transcriptional apparatus in the form of the RNA polymerase II holoenzyme.

Chromatin↗
PubMed1996

A new class of activation-defective TATA-binding protein mutants: evidence for two steps of transcriptional activation in vivo.

Using a genetic screen, we isolated four TATA-binding protein (TBP) mutants that are specifically defective in vivo for the response to acidic activators. In contrast to previously described activation-defective TBP mutants, these TBP derivatives are not specifically defective for interactions with TATA elements or TFIIA. Three of these derivatives interact normally with a TATA element, TFIIA, TFIIB, or an acidic activation domain; presumably, they affect another protein-protein interaction important for transcriptional activation. The remaining derivative (with F-237 replaced by D) binds a TATA element with wild-type affinity, but the TBP-TATA complex has an altered electrophoretic mobility and interacts poorly with TFIIA and TFIIB; this suggests that the conformation of the TBP-TATA element complex plays a role in transcriptional activation. To determine the step at which the TBP derivatives were unable to activate transcription, we utilized an artificial recruitment assay in which TBP is targeted to the promoter via fusion to the LexA DNA-binding domain. Consistent with previous evidence that acidic activators can increase recruitment of TBP to the promoter in vivo, the activation defect of some of these TBP derivatives can be corrected by artificial recruitment. In contrast, the activation defect of the other TBP derivatives is not bypassed by artificial recruitment. Thus, these TBP mutants define two steps in the process of transcriptional stimulation by acidic activators: efficient recruitment to the TATA element and a postrecruitment interaction with a component(s) of the initiation complex.

DNA-Binding Proteins↗
PubMed1995

Determinants of half-site spacing preferences that distinguish AP-1 and ATF/CREB bZIP domains.

The AP-1 and ATF/CREB families of eukaryotic transcription factors are dimeric DNA-binding proteins that contain the bZIP structural motif. The AP-1 and ATF/CREB proteins are structurally related and recognize identical half-sites (TGAC), but they differ in their requirements for half-site spacing. AP-1 proteins such as yeast GCN4 preferentially bind to sequences with overlapping half-sites, whereas ATF/CREB proteins bind exclusively to sequences with adjacent half-sites. Here we investigate the distinctions between AP-1 and ATF/CREB proteins by determining the DNA-binding properties of mutant and hybrid proteins. First, analysis of GCN4-ATF1 hybrid proteins indicates that a short surface spanning the basic and fork regions of the bZIP domain is the major determinant of half-site spacing. Replacement of two GCN4 residues on this surface (Ala244 and Leu247) by their ATF1 counterparts largely converts GCN4 into a protein with ATF/CREB specificity. Secondly, analysis of a Fos derivative containing the GCN4 leucine zipper indicates that Fos represents a novel intermediate between AP-1 and ATF/CREB proteins. Thirdly, we examine the effects of mutations in the invariant arginine residue of GCN4 (Arg243) that contacts the central base pair(s) of the target sites. While most mutations abolish DNA binding, substitution of a histidine residue results in a GCN4 derivative with ATF/CREB binding specificity. These results suggest that the AP-1 and ATF/CREB proteins differ in positioning a short surface that includes the invariant arginine and that AP-1 proteins may represent a subclass (and perhaps evolutionary offshoot) of ATF/CREB proteins that can tolerate overlapping half-sites.

Activating Transcription Factors↗
PubMed1995

The TBP-TFIIA interaction in the response to acidic activators in vivo.

A yeast TBP mutant (N2-1) is described here that is defective specifically in responding to acidic activators in vivo. N2-1 does not support activation by Gal4, Ace1, and Gcn4, but appears unaffected for constitutive transcription, repression by the Cyc8-Tup1 and Not complexes, and transcription by polymerase I (Pol) and Pol III. In vitro, N2-1 fails to interact with TFIIA, but it associates normally with a TATA element, an acidic activation domain, and TFIIB. Fusion of the small subunit of TFIIA to N2-1 restores activation function in vivo. Thus, an efficient interaction between TBP and TFIIA is required for transcriptional activation in vivo.

Base Sequence↗
PubMed1995

Poly(dA:dT), a ubiquitous promoter element that stimulates transcription via its intrinsic DNA structure.

Many yeast promoters contain homopolymeric dA:dT sequences that affect nucleosome formation in vitro and are required for wild-type levels of transcription in vivo. Here, we show that poly(dA:dT) is a novel promoter element whose function depends on its intrinsic structure, not its interaction with sequence-specific, DNA-binding proteins. First, poly(dA:dT) stimulates Gcn4-activated transcription in a manner that is length dependent and inversely related to intracellular Gcn4 levels. Second, Datin, the only known poly(dA:dT)-binding protein, behaves as a repressor through poly(dA:dT) sequences. Third, poly(dG:dC), a structurally dissimilar homopolymer that also affects nucleosomes, has transcriptional properties virtually identical to those of poly(dA:dT). Three probes of chromatin structure including HinfI endonuclease cleavage in vivo indicate that poly(dA:dT) increases accessibility of the Gcn4 binding site and adjacent sequences in physiological chromatin. These observations suggest that, by virtue of its intrinsic structure, poly(dA:dT) locally affects nucleosomes and increases the accessibility of transcription factors bound to nearby sequences.

Base Sequence↗
PubMed1995

Connecting a promoter-bound protein to TBP bypasses the need for a transcriptional activation domain.

Biochemical analyses have suggested potential targets for transcriptional activation domains, which include several components of the RNA polymerase II machinery, as well as the chromatin template. Here we examine the mechanism of transcriptional activation in yeast cells by connecting a heterologous DNA-binding domain (LexA) to the TATA-binding protein (TBP). LexA-TBP efficiently activates transcription from a promoter containing a LexA operator upstream of a TATA element. Activation is promoter-specific and is sensitive to mutations on the DNA-binding surface of TBP; hence it is not due to a fortuitous activation domain on TBP. Thus a promoter-bound protein lacking an activation domain can stimulate transcription if it is directly connected to TBP. This suggests that recruitment of TBP to the promoter can be a rate-limiting step for transcription in vivo, and that interactions between activation domains and factors that function after TBP recruitment can be bypassed for activation.

Bacterial Proteins↗
PubMed1995

Distinct TPR motifs of Cyc8 are involved in recruiting the Cyc8-Tup1 corepressor complex to differentially regulated promoters.

The yeast Cyc8(Ssn6)-Tup1 complex is required for transcriptional repression of distinct sets of genes that are regulated by glucose, oxygen, cell type, and DNA damage. It has been proposed that the Cyc8-Tup1 complex is a corepressor that is recruited to promoters by interacting with pathway-specific DNA-binding proteins. Previously, we showed that a specific region of Tup1 mediates the general transcriptional repression function of the complex. Here, we define functional domains of Cyc8, a protein consisting primarily of 10 tandem copies of a TPR motif. Distinct combinations of TPR motifs are required specifically for direct interaction with Tup1, repression of oxygen-regulated genes, and repression of glucose-regulated genes. In contrast, the WD motifs of Tup1 are not essential for repression of genes regulated by glucose and oxygen, but they are required for those regulated by cell type and DNA damage. In addition, we show that the Cyc8-Tup1 complex functions both as a corepressor and an inhibitor of Mig1, a protein that binds to promoters of glucose-repressible genes. These observations suggest that different Cyc8 TPR motifs and the Tup1 WD domain mediate distinct protein-protein interactions that link the Cyc8-Tup1 corepressor to structurally dissimilar DNA-binding proteins required for pathway-specific regulation.

Binding Sites↗
PubMed1995

Mutations on the DNA-binding surface of TATA-binding protein can specifically impair the response to acidic activators in vivo.

The TATA-binding protein (TBP) contains a concave surface that interacts specifically with TATA promoter elements and a convex surface that mediates protein-protein interactions with general and gene-specific transcription factors. Biochemical experiments suggest that interactions between activator proteins and TBP are important in stimulating transcription by the RNA polymerase II machinery. To gain insight into the role of TBP in mediating transcriptional activation in vivo, we implemented a genetic strategy in Saccharomyces cerevisiae that involved the use of a TBP derivative with altered specificity for TATA elements. By genetically screening a set of TBP mutant libraries that were biased to the convex surface that mediates protein-protein interactions, we identified TBP derivatives that are impaired in the response to three acidic activators (Gcn4, Gal4, and Ace1) but appear normal for constitutive polymerase II transcription. A genetic complementation assay indicates that the activation-defective phenotypes reflect specific functional properties of the TBP derivatives rather than an indirect effect on transcription. Surprisingly, three of the four activation-defective mutants affect residues that directly contact DNA. Moreover, all four mutants are defective for TATA element binding, but they interact normally with an acidic activation domain and TFIIB. In addition, we show that a subset of TBP derivatives with mutations on the DNA-binding surface of TBP are also compromised in their responses to acidic activators in vivo. These observations suggest that interactions at the TBP-TATA element interface can specifically affect the response to acidic activator proteins in vivo.

Base Sequence↗
PubMed1995

Mechanism of differential utilization of the his3 TR and TC TATA elements.

The yeast his3 promoter region contains two TATA elements, TC and TR, that are differentially utilized in constitutive his3 transcription and Gcn4-activated his3 transcription. TR contains the canonical TATAAA sequence, whereas TC is an extended region that lacks a conventional TATA sequence and does not support transcription in vitro. Surprisingly, differential his3 TATA-element utilization does not depend on specific properties of activator proteins but, rather, is determined by the overall level of his3 transcription. At low levels of transcription, the upstream TC is preferentially utilized, even though it is inherently a much weaker TATA element than TR. The TATA elements are utilized equally at intermediate levels, whereas TR is strongly preferred at high levels of transcription. This characteristic behavior can be recreated by replacing TC with moderately functional derivatives of a conventional TATA element, suggesting that TC is a collection of weak TATA elements. Analysis of promoters containing two biochemically defined TATA elements indicates that differential utilization occurs when the upstream TATA element is weaker than the downstream element. In other situations, the upstream TATA element is preferentially utilized in a manner that is independent of the overall level of transcription. Thus, in promoters containing multiple TATA elements, relative utilization not only depends on the quality and arrangement of the TATA elements but can vary with the overall level of transcriptional stimulation. We suggest that differential TATA utilization results from the combination of an intrinsic preference for the upstream element and functional saturation of weak TATA elements at low levels of transcriptional stimulation.

Base Sequence↗
PubMed1995

The glutamine-rich activation domains of human Sp1 do not stimulate transcription in Saccharomyces cerevisiae.

Eukaryotic transcriptional activators have been classified on the basis of the characteristics of their activation domains. Acidic activation domains, such as those in the yeast GAL4 or GNC4 proteins and the herpes simplex virus activator VP16, stimulate RNA polymerase II transcription when introduced into a variety of eukaryotic cells. This species interchangeability demonstrates that the mechanism by which acidic activation domains function is highly conserved in the eukaryotic kingdom. To determine whether such a conservation of function exists for a different class of activation domain, we have tested whether the glutamine-rich activation domains of the human transcriptional activator Sp1 function in the yeast Saccharomyces cerevisiae. We report here that the glutamine-rich domains of Sp1 do not stimulate transcription in S. cerevisiae, even when accompanied by human TATA-box binding protein (TBP) or human-yeast TATA-box binding protein hybrids. Thus, in contrast to the case for acidic activation domains, the mechanism by which glutamine-rich domains stimulate transcription is not conserved between S. cerevisiae and humans.

Amino Acid Sequence↗
PubMed1995

Yeast transcriptional regulatory mechanisms.

Transcriptional regulation directly influences many biological phenomena such as cell growth, response to environmental change, development of multicellular organisms, and disease. Transcriptional regulatory mechanisms are fundamentally similar in eukaryotic organisms (93). Components of the basic RNA polymerase II (Pol II) machinery are highly conserved and, in some cases, functionally interchangeable. Transcription factors with similar structures and DNA-binding specificities are found throughout the eukaryotic kingdom, and acidic activation domains stimulate transcription across a wide range of species. Complex promoters with multiple protein binding sites are typical in all eukaryotic organisms, and efficient transcription generally requires the combinatorial and synergistic action of activator proteins that function at long and variable distances from the mRNA initiation site. Molecular mechanisms of eukaryotic transcriptional regulation have been elucidated from the studies that involve a wide variety of genes, promoters, proteins, organisms, and experimental approaches. This review focuses on transcriptional regulatory mechanisms in the baker's yeast Saccharomyces cerevisiae. Studies in yeast have emphasized powerful genetic approaches that are not available in other eukaryotic organisms. As a consequence, yeast is particularly amenable for analyzing transcriptional regulatory mechanisms in vivo under true physiological conditions. Furthermore, classical and molecular yeast genetics has permitted the discovery and functional characterization of transcriptional regulatory proteins that were not identified in biochemical studies. Thus, genetic analysis in yeast has often generated information complementary to that obtained from biochemical studies of transcription in vitro, and it has provided unique insights into mechanisms of eukaryotic transcriptional regulation.

Animals↗
PubMed1994

Increased recruitment of TATA-binding protein to the promoter by transcriptional activation domains in vivo.

The rate at which the TATA-binding protein (TBP) interacts with the TATA element and promotes transcription by RNA polymerase II was determined in yeast cells. A TBP derivative with altered TATA-element specificity was rapidly induced, and transcription from promoters with appropriately mutated TATA elements was measured. Without a functional activator protein, basal transcription was observed only after a lag of several hours. In contrast, GCN4-activated transcription occurred rapidly upon induction of the TBP derivative. These results suggest that accessibility of TBP to the chromatin template in vivo is rate limiting and that activation domains increase recruitment of TBP to the promoter.

Base Sequence↗