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K Struhl

Publications and source records attributed to K Struhl.

At least 91 records · Page 5Linked to original sources
PubMed1992

Yeast and human TFIID with altered DNA-binding specificity for TATA elements.

TFIID is the highly conserved, but species-specific, component of the RNA polymerase II transcription machinery that binds specifically to the TATA element (consensus TATAAA). Using a genetic selection, we isolated an altered specificity derivative of yeast TFIID that permits transcription from promoters containing a mutated TATA element (TGTAAA). Biochemical analysis indicates that this TFIID derivative has specifically gained the ability to bind TGTAAA efficiently. The mutant protein contains three substitutions within a 12 amino acid region; two of these are necessary and primarily responsible for the altered specificity. An analogous version of human TFIID, generated by introducing the same amino acid substitutions in the corresponding region of the protein, can support basal and GCN4-activated transcription in yeast cells from a TGTAAA-containing promoter. These results define a surface of TFIID that directly interacts with the TATA element, and they indicate that human TFIID can respond to acidic activator proteins in conjunction with the other components of the yeast transcription machinery.

Amino Acid Sequence↗
PubMed1992

Yeast and human TFIIDs are interchangeable for the response to acidic transcriptional activators in vitro.

Previous work showed that human TFIID fails to support yeast cell growth, although it is nearly identical to yeast TFIID in a carboxy-terminal region of the molecule that suffices for basal, TATA-element-dependent transcription in vitro. These and other findings raised the possibility that TFIID participates in species-specific interactions, possibly with mediator factors, required for activated transcription. Here, we report that human TFIID and amino-terminally truncated derivatives of yeast TFIID are fully functional in support of both basal transcription and the response to acidic activator proteins in a yeast in vitro transcription system. Conversely, and in contrast to previously published results, yeast TFIID supports both basal and activated transcription in reactions reconstituted with human components. This functional interchangeability of yeast and human TFIIDs argues strongly against species specificity with regard to TFIID function in basal transcription and the response to acidic activator proteins. In addition, our results suggest that any intermediary factors between acidic activators and TFIID are conserved from yeast to man.

Blotting, Northern↗
PubMed1992

Yeast GCN4 as a probe for oncogenesis by AP-1 transcription factors: transcriptional activation through AP-1 sites is not sufficient for cellular transformation.

The Jun and Fos oncoproteins belong to the AP-1 family of transcriptional activators and are believed to induce cellular transformation by inappropriately activating genes involved in cell replication. To determine whether transcriptional activation through AP-1 sites is sufficient for transforming activity, we examined the properties of an autonomous and heterologous AP-1 protein, yeast GCN4, in rat embryo fibroblasts. GCN4 induces transcriptional activation through AP-1 sites but, unlike Jun and Fos, fails to induce cellular transformation, in cooperation with Ha-ras. Jun-GCN4 and Fos-GCN4 homodimers independently induce cellular transformation indicating that the amino-terminal regions of Jun and Fos each contain regulatory functions that are required for oncogenesis but are distinct from generic transcriptional activation domains. In addition, these observations have implications for the nature of the oncogenically relevant target genes that respond to Jun and Fos.

Base Sequence↗
PubMed1992

ACR1, a yeast ATF/CREB repressor.

Members of the mammalian ATF/CREB family of transcription factors, which are associated with regulation by cyclic AMP and viral oncogenes, bind common DNA sequences (consensus TGACGTCA) via a bZIP domain. In the yeast Saccharomyces cerevisiae, ATF/CREB-like sequences confer either repression or activation of transcription, depending on the promoter context. By isolating mutations that alleviate the repression mediated by ATF/CREB sites, we define a new yeast gene, ACR1, which encodes an ATF/CREB transcriptional repressor. ACR1 contains a bZIP domain that is necessary for homodimer formation and specific DNA binding to an ATF/CREB site. Within the bZIP domain, ACR1 most strongly resembles the mammalian cyclic AMP-responsive transcriptional regulators CREB and CREM; it is less similar to GCN4 and YAP1, two previously described yeast bZIP transcriptional activators that recognize the related AP-1 sequence (consensus TGACTCA). Interestingly, deletion of the ACR1 gene causes increased transcription through ATF/CREB sites that does not depend on GCN4 or YAP1. Moreover, extracts from acr1 deletion strains contain one or more ATF/CREB-like DNA-binding activities. These genetic and biochemical observations suggest that S. cerevisiae contains a family of ATF/CREB proteins that function as transcriptional repressors or activators.

Amino Acid Sequence↗
PubMed1991

The leucine zipper symmetrically positions the adjacent basic regions for specific DNA binding.

The bZIP structural motif present in several eukaryotic transcription factors is defined by the leucine zipper, a coiled-coil dimerization interface, and an adjacent basic region that directly interacts with DNA. To examine the functional importance of the highly conserved spacing between the leucine zipper and the basic region, we have analyzed the DNA-binding ability of yeast GCN4 proteins containing amino acid insertions between these two subdomains. Proteins containing a surprisingly wide variety of seven-amino acid insertions, but none containing two-, four-, or six-amino acid insertions, are functional. However, heterodimers between wild-type GCN4 and functional derivatives containing seven amino acid insertions are unable to bind DNA. These observations provide strong experimental support for several aspects of the scissors grip and induced fork models for DNA-binding by bZIP proteins. Specifically, they demonstrate that continuous alpha-helices symmetrically diverging from the leucine zipper correctly position the two basic regions for specific binding to abutting DNA half-sites. In addition, the results indicate that GCN4 homodimers are primarily responsible for transcriptional activation in yeast cells.

Amino Acid Sequence↗
PubMed1991

Functional differences between yeast and human TFIID are localized to the highly conserved region.

TFIID, the general transcription factor that binds TATA promoter elements, is highly conserved throughout the eukaryotic kingdom. TFIIDs from different organisms contain C-terminal core domains that are at least 80% identical and display similar biochemical properties. Despite these similarities, yeast cells containing human TFIID instead of the endogenous yeast protein grow extremely poorly. Surprisingly, this functional distinction reflects differences in the core domains, not the divergent N-terminal regions. The N-terminal region is unimportant for the essential function(s) of yeast TFIID because expression of the core domain permits efficient cell growth. Analysis of yeast-human hybrid TFIIDs indicates that several regions within the conserved core account for the phenotypic difference, with some regions being more important than others. This species specificity might reflect differences in DNA-binding properties and/or interactions with activator proteins or other components of the RNA polymerase II transcription machinery.

Base Sequence↗
PubMed1991

Synergistic transcriptional enhancement does not depend on the number of acidic activation domains bound to the promoter.

Many eukaryotic transcriptional activator proteins contain a DNA-binding domain that interacts with specific promoter sequences and an acidic activation region that is required to stimulate transcription. Transcriptional enhancement by such activator proteins is often synergistic and promiscuous; promoters containing multiple binding sites for an individual protein or even for unrelated proteins can be 10-100 times more active than promoters with single sites. It has been suggested that such synergy reflects a nonlinear response of the basic transcription machinery to the number and/or quality of acidic activation regions. Here, we determine the transcriptional activity of Jun-Fos heterodimers containing one or two GCN4 acidic activation regions on promoters containing one or two Ap-1 target sites. Surprisingly, heterodimers with one or two acidic regions activate transcription with similar efficiency and are equally synergistic (10- to 15-fold) on promoters containing two target sites. Thus, transcriptional synergy does not depend on the number of acidic activation regions but rather on the number of proteins bound to the promoter. This suggests that synergy is mediated either by cooperative DNA binding or by alternative mechanisms in which the DNA-binding domain plays a more direct role in transcription (e.g., changes in DNA structure, nucleosome displacement, or direct interactions with the transcriptional machinery).

Base Sequence↗
PubMed1991

Mechanisms for diversity in gene expression patterns.

Despite the relatively low number of transcriptional regulatory proteins, the number of possible combinations that act in particular cell types at specific times and in response to appropriate extracellular stimuli is enormous. In considering the regulatory patterns of a particular gene, the critical determinants of diversity are the specific promoter sequences that govern the potential DNA-binding proteins which function either directly or indirectly in association with other proteins; constellations of proteins in the nucleus and their transcriptional activities; and synergistic or antagonistic protein-protein interactions. Although some of these regulatory principles operate in prokaryotes, the combinatorial nature of the transcriptional activation process, the existence of multiprotein families, and the prevalence of heteromeric protein complexes are characteristic of eukaryotic cells and are essential for the extraordinary complexity of gene expression patterns in multicellular organisms.

Gene Expression Regulation↗
PubMed1991

Highly conserved residues in the bZIP domain of yeast GCN4 are not essential for DNA binding.

Yeast GCN4 and the Jun oncoprotein are transcriptional activators that bind DNA via a bZIP domain consisting of a leucine zipper dimerization element and an adjacent basic region that directly contacts DNA. Two highly conserved alanines (Ala-238 and Ala-239 in GCN4) and an invariant asparagine (Asn-235) in the basic region have been proposed to play important roles in DNA sequence recognition by bZIP proteins. Surprisingly, these conserved residues can be functionally replaced in GCN4 and in a derivative containing the Jun basic region (Jun-GCN4). The ability of an amino acid to functionally substitute for Asn-235 does not correlate with its preference for assuming the N-cap position of an alpha helix. This finding argues against the proposal of the scissors grip model that the invariant asparagine forms an N cap that permits the basic region to bend sharply and wrap around the DNA. In contrast to a prediction of the induced fork model, the pattern of functional substitutions of the conserved alanines together with the results of uracil interference experiments suggests that Ala-238 and Ala-239 do not make base-specific DNA contacts. Finally, the Jun-GCN4 chimeric proteins appear much more active in vivo than expected from their DNA-binding properties in vitro. The mechanistic and evolutionary implications of these results are discussed.

Alanine↗
PubMed1990

Folding transition in the DNA-binding domain of GCN4 on specific binding to DNA.

Protein-DNA recognition is often mediated by a small domain containing a recognizable structural motif, such as the helix-turn-helix or the zinc-finger. These motifs are compact structures that dock against the DNA double helix. Another DNA recognition motif, found in a highly conserved family of eukaryotic transcription factors including C/EPB, Fos, Jun and CREB, consists of a coiled-coil dimerization element the leucine-zipper and an adjoining basic region which mediates DNA binding. Here we describe circular dichroism and 1H-NMR spectroscopic studies of another family member, the yeast transcriptional activator GCN4. The 58-residue DNA-binding domain of GCN4, GCN4-p, exhibits a concentration-dependent alpha-helical transition, in accord with previous studies of the dimerization properties of an isolated leucine-zipper peptide. The GCN4-p dimer is approximately 70% helical at 25 degrees C, implying that the basic region adjacent to the leucine zipper is largely unstructured in the absence of DNA. Strikingly, addition of DNA containing a GCN4 binding site (AP-1 site) increases the alpha-helix content of GNC4-p to at least 95%. Thus, the basic region acquires substantial alpha-helical structure when it binds to DNA. A similar folding transition is observed on GCN4-p binding to the related ATF/CREB site, which contains an additional central base pair. The accommodation of DNA target sites of different lengths clearly requires some flexibility in the GCN4 binding domain, despite its high alpha-helix content. Our results indicate that the GCN4 basic region is significantly unfolded at 25 degrees C and that its folded, alpha-helical conformation is stabilized by binding to DNA.

Activating Transcription Factors↗
PubMed1990

A wide variety of DNA sequences can functionally replace a yeast TATA element for transcriptional activation.

We created a library of DNA molecules in which the required TATA element of a yeast gal-his3 promoter is replaced by random-sequence oligomers averaging 16 bp in length. Surprisingly, 1% of such random sequences functionally replace the native yeast TATA element. In many cases, sequences completely unrelated to the consensus TATA element (TATAAA) stimulate transcription with equal or increased efficiency. Transcription mediated by these synthetic elements requires GAL4 and is initiated from normal his3 initiation sites, suggesting that it occurs by a mechanism indistinguishable from that involving wild-type TATA elements. Many, but not all, of these elements act as substrates for yeast TFIID in reconstituted transcription reactions in vitro. These observations indicate that yeast TFIID can stimulate transcription from TATA elements whose sequences differ from the consensus, and they suggest the possibility of alternative factors that may provide a related function for transcriptional activation.

Animals↗
PubMed1990

Mutations that define the optimal half-site for binding yeast GCN4 activator protein and identify an ATF/CREB-like repressor that recognizes similar DNA sites.

The yeast GCN4 transcriptional activator protein binds as a dimer to a dyad-symmetric sequence, indicative of a protein-DNA complex in which two protein monomers interact with adjacent half-sites. However, the optimal GCN4 recognition site, ATGA(C/G)TCAT, is inherently asymmetric because it contains an odd number of base pairs and because mutation of the central C.G base pair strongly reduces specific DNA binding. From this asymmetry, we suggested previously that GCN4 interacts with nonequivalent and possibly overlapping half-sites (ATGAC and ATGAG) that have different affinities. Here, we examine the nature of GCN4 half-sites by creating symmetrical derivatives of the optimal GCN4 binding sequence that delete or insert a single base pair at the center of the site. In vitro, GCN4 bound efficiently to the sequence ATGACGTCAT, whereas it failed to bind to ATGAGCTCAT or ATGATCAT. These observations strongly suggest that (i) GCN4 specifically recognizes the central base pair, (ii) the optimal half-site for GCN4 binding is ATGAC, not ATGAG, and (iii) GCN4 is a surprisingly flexible protein that can accommodate the insertion of a single base pair in the center of its compact binding site. The ATGACGTCAT sequence strongly resembles sites bound by the yeast and mammalian ATF/CREB family of proteins, suggesting that GCN4 and the ATF/CREB proteins recognize similar half-sites but have different spacing requirements. Unexpectedly, in the context of the his3 promoter, the ATGACGTCAT derivative reduced transcription below the basal level in a GCN4-independent manner, presumably reflecting DNA binding by a distinct ATF/CREB-like repressor protein. In other promoter contexts, however, the same site acted as a weak upstream activating sequence.

Base Sequence↗
PubMed1990

Analysis of Saccharomyces cerevisiae his3 transcription in vitro: biochemical support for multiple mechanisms of transcription.

The promoter region of the Saccharomyces cerevisiae his3 gene contains two TATA elements, TC and TR, that direct transcription initiation to two sites designated +1 and +13. On the basis of differences between their nucleotide sequences and their responsiveness to upstream promoter elements, it has previously been proposed that TC and TR promote transcription by different molecular mechanisms. To begin a study of his3 transcription in vitro, we used S. cerevisiae nuclear extracts together with various DNA templates and transcriptional activator proteins that have been characterized in vivo. We demonstrated accurate transcription initiation in vitro at the sites used in vivo, transcriptional activation by GCN4, and activation by a GAL4 derivative on various gal-his3 hybrid promoters. In all cases, transcription stimulation was dependent on the presence of an acidic activation region in the activator protein. In addition, analysis of promoters containing a variety of TR derivatives indicated that the level of transcription in vitro was directly related to the level achieved in vivo. The results demonstrated that the in vitro system accurately reproduced all known aspects of in vivo his3 transcription that depend on the TR element. However, in striking contrast to his3 transcription in vivo, transcription in vitro yielded approximately 20 times more of the +13 transcript than the +1 transcript. This result was not due to inability of the +1 initiation site to be efficiently utilized in vitro, but rather it reflects the lack of TC function in vitro. The results support the idea that TC and TR mediate transcription from the wild-type promoter by distinct mechanisms.

Base Sequence↗
PubMed1990

Yeast and human TATA-binding proteins have nearly identical DNA sequence requirements for transcription in vitro.

We have analyzed the DNA sequence requirements for TATA element function by assaying the transcriptional activities of 25 promoters, including those representing each of the 18 single-point mutants of the consensus sequence TATAAA, in a reconstituted in vitro system that depends on the TATA element-binding factor TFIID. Interestingly, yeast TFIID and HeLa cell TFIID were virtually identical in terms of their relative activities on this set of promoters. Of the mutated elements, only two had undetectable activity; the rest had activities ranging from 2 to 75% of the activity of the consensus element, which was the most active. In addition, mutations of the nucleotide following the TATAAA core strongly influenced transcriptional activity, although with somewhat different effects on yeast and HeLa TFIID. The activities of all these promoters depended upon TFIID, and the level of TFIID-dependent transcription in vitro correlated strongly with their activities in yeast cells. This suggests that the in vivo activities of these elements reflect their ability to functionally interact with a single TATA-binding factor. However, some elements with similar activities in vitro supported very different levels of transcriptional activation by GAL4 protein in vivo. These results extend the degree of evolutionary conservation between yeast and mammalian TFIID and are useful for predicting the level of TATA element function from the primary sequence.

Base Sequence↗
PubMed1990

A nucleosome-positioning sequence is required for GCN4 to activate transcription in the absence of a TATA element.

In the gal-his3 hybrid promoter his3-GG1, the yeast upstream activator protein GCN4 stimulates transcription when bound at the position normally occupied by the TATA element. This TATA-independent activation by GCN4 requires two additional elements in the gal enhancer region that are distinct from those involved in normal galactose induction. Both additional elements appear to be functionally distinct from a classical TATA element because they cannot be replaced by the TFIID-binding sequence TATAAA. One of these elements, termed Q, is essential for GCN4-activated transcription and contains the sequence GTCAC CCG, which overlaps (but is distinct from) a GAL4 binding site. Surprisingly, relatively small increases in the distance between Q and the GCN4 binding site significantly reduce the level of transcription. The Q element specifically interacts with a yeast protein (Q-binding protein [QBP]) that may be equivalent to Y, a protein that binds at a sequence that forms a constraint to nucleosome positioning. Analysis of various deletion mutants indicates that the sequence requirements for binding by QBP in vitro are indistinguishable from those necessary for Q activity in vivo, strongly suggesting that QBP is required for the function of this TATA-independent promoter. These results support the view that transcriptional activation can occur by an alternative mechanism in which the TATA-binding factor TFIID either is not required or is not directly bound to DNA. In addition, they suggest a potential role of nucleosome positioning for the activity of a promoter.

Alleles↗
PubMed1990

Tc, an unusual promoter element required for constitutive transcription of the yeast HIS3 gene.

Tc is the proximal promoter element required for constitutive his3 transcription that occurs in the absence of the canonical TATA element (TR) and is initiated from the +1 site. The TC element, unlike TR, does not respond to transcriptional stimulation by the GCN4 or GAL4 activator protein. Analysis of deletion, substitution, and point mutations indicates that Tc mapped between nucleotides -54 and -83 and is a sequence-dependent element because it could not be functionally replaced by other DNA sequences. However, in contrast to the behavior of typical promoter elements, it was surprisingly difficult to eliminate Tc function by base pair substitutions. Of 15 derivatives averaging four substitutions in the Tc region and representing 40% of all possible single changes, only 1 inactivated the Tc element. Moreover, the phenotypes of mutant and hybrid elements indicated that inactivation of Tc required multiple changes. The spacing between Tc and the initiation region could be varied over a 30-base-pair range without significantly affecting the level of transcription from the +1 site. From these results, we consider it possible that Tc may not interact with TFIID or some other typical sequence-specific transcription factor, but instead might influence transcription, either directly or indirectly, by its DNA structure.

Base Sequence↗