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K Struhl

Publications and source records attributed to K Struhl.

At least 109 records · Page 6Linked to original sources
PubMed1989

Changing fos oncoprotein to a jun-independent DNA binding protein with GCN4 dimerization specificity by swapping "leucine zippers".

A structural motif for DNA-binding proteins, the 'leucine zipper', has been proposed for the jun, fos and myc gene products, the yeast transcriptional activator GCN4, and the C/EBP enhancer-binding protein. These proteins all contain a region with four or five leucine residues spaced exactly seven amino acid residues apart whose sequence is consistent with the formation of an amphipathic alpha-helix. It has been proposed that the leucine zipper consists of two interdigitated alpha-helices, one from each monomer, that constitute the dimerization function necessary for high-affinity binding to DNA; an adjacent region of basic residues is thought to be responsible for specific protein-DNA contacts. In support of this model, substitution of the leucine residues within the motif can abolish dimerization and DNA-binding, and a synthetic peptide corresponding to the GCN4 leucine zipper forms alpha-helical dimers. Despite the conserved leucine residues, however, each protein has a distinct dimerization specificity. Specifically, GCN4 homodimer, Jun homodimer and Fos-Jun heterodimer proteins bind to the same DNA site, whereas Fos is unable to form homodimers, bind DNA, or interact with GCN4 (refs 8-14). Here, we alter the dimerization specificity of Fos by precisely replacing its leucine zipper with that from GCN4. This Fos-GCN4 chimaeric protein is able to bind to the target site in the absence of Jun, and can form DNA-binding heterodimers with GCN4 but not with Jun. These results indicate that the leucine zipper is sufficient to confer dimerization specificity and strongly suggest that Fos contacts DNA directly.

Binding Sites↗
PubMed1989

The gradient morphogen bicoid is a concentration-dependent transcriptional activator.

The bicoid (bcd) protein is expressed in an anteroposterior gradient in early Drosophila embryos and controls the zygotic activation of the segmentation gene hunchback (hb) in a broad but precisely bounded anterior domain. Here we show that the hb gene contains multiple regulatory elements that mediate transcriptional activation in response to bcd protein. Further, we demonstrate that the resulting patterns of expression in vivo depend critically on both the bcd gradient profile and the number and quality of these hb elements. Finally, we show that these same elements mediate bcd-dependent transcriptional activation in yeast and that this interaction requires distinct DNA binding and activating regions in the bcd protein. Our results argue that bcd protein normally binds and activates the hb gene in a concentration-dependent fashion, thereby allowing the gradient of bcd protein to dictate where the hb gene is initially turned on in early embryos. They also suggest that the bcd gradient has the instructive capacity to activate other subordinate control genes by the same mechanism, each in a distinct spatial domain according to its affinity for bcd protein.

Animals↗
PubMed1989

Yeast upstream activator protein GCN4 can stimulate transcription when its binding site replaces the TATA element.

We replaced the required TATA element of a yeast gal-his3 promoter by a binding site for GCN4, a protein that normally activates transcription when bound upstream of a TATA element. Surprisingly, GCN4 efficiently activates his3 transcription from wild-type initiation sites, though in a pattern associated with constitutive his3 transcription rather than GCN4 upstream activation through a TATA element. Transcriptional stimulation by GCN4 requires both the DNA-binding domain and the acidic activation function but is not affected by changing the spacing or helical relationship between the GCN4 binding site and the mRNA start sites. GCN4 is not sufficient for this TATA-independent activation; a sequence in the gal fragment distinct from the GAL4 binding sites is also required. Thus, GCN4 functions both when bound upstream of a TATA element and also when bound at the position of a TATA element. In the latter case, we suggest the possibility that GCN4 might be able to stimulate transcription by an alternate mechanism that does not involve a conventional TATA-binding transcription factor.

Bacterial Proteins↗
PubMed1989

Helix-turn-helix, zinc-finger, and leucine-zipper motifs for eukaryotic transcriptional regulatory proteins.

Four distinct structural motifs have been proposed for the DNA-binding domains of eukaryotic transcriptional regulatory proteins; the helix-turn-helix, two kinds of zinc finger, and the leucine zipper. Within each structural motif, there are often families of related proteins that recognize similar DNA sequences and are conserved throughout the eukaryotic kingdom. However, the processes of transcriptional activation and repression appear to be independent of the specific type of protein-DNA interaction.

Base Sequence↗
PubMed1989

An efficient method for generating proteins with altered enzymatic properties: application to beta-lactamase.

Random-sequence or highly degenerate oligonucleotides have been useful for defining functionally important sequences both in proteins and in nucleic acids. In this approach, such oligonucleotides are used to replace a segment of DNA required for a desired function, and functional sequences are identified by an appropriate genetic or biochemical selection. Here, a collection of 500,000 [corrected] altered beta-lactamase proteins was generated by cloning a mixed-base oligonucleotide in place of the sequences coding for a 17-amino acid portion of the enzyme's active site. Approximately 2000 enzymes from this collection were able to confer ampicillin resistance on Escherichia coli. Fifty-eight of these were chosen for further study after characterization with various beta-lactam substrates. beta-Lactamases having altered specificity against different antibiotics, resistance to the suicide inhibitors clavulanic acid and sulbactam, and temperature-dependent activities were obtained. The amino acid residues responsible for these altered properties as well as for basic enzyme activity are defined. This approach should prove to be an effective and general tool for creating proteins with novel properties, especially in situations in which a high-resolution structure of the protein is not known.

Amino Acid Sequence↗
PubMed1989

Yeast GCN4 transcriptional activator protein interacts with RNA polymerase II in vitro.

Regulated transcription by eukaryotic RNA polymerase II (Pol II) requires the functional interaction of multiple protein factors, some of which presumably interact directly with the polymerase. One such factor, the yeast GCN4 activator protein, binds to the upstream promoter elements of many amino acid biosynthetic genes and induces their transcription. Through the use of affinity chromatography involving GCN4- or Pol II-Sepharose columns, we show that GCN4 interacts specifically with Pol II in vitro. Purified Pol II is retained on the GCN4-Sepharose column under conditions in which the vast majority of proteins flow through. Moreover, Pol II can be selectively isolated from more complex mixtures of proteins. Conversely, GCN4 protein, synthesized in vitro or in Escherichia coli, specifically binds to the Pol II-Sepharose column under equivalent conditions. Using deletion mutants, we also show that the DNA-binding domain of GCN4 is both necessary and sufficient for this interaction. We suggest the possibility that this GCN4-Pol II interaction may be important for transcription in vivo.

Binding Sites↗
PubMed1989

Functional distinctions between yeast TATA elements.

Although the yeast his3 promoter region contains two functional TATA elements, TR and TC, the GCN4 and GAL4 upstream activator proteins stimulate transcription only through TR. In combination with GAL4, an oligonucleotide containing the sequence TATAAA is fully sufficient for TR function, whereas almost all single-base-pair substitutions of this sequence abolish the ability of this element to activate transcription. Further analysis of these and other mutations of the TR element led to the following conclusions. First, sequences downstream of the TATAAA sequence are important for TR function. Second, a double mutant, TATTTA, can serve as a TR element even though the corresponding single mutation, TATTAA, is unable to do so. Third, three mutations have the novel property of being able to activate transcription in combination with GCN4 but not with GAL4; this finding suggests that activation by GCN4 and by GAL4 may not occur by identical mechanisms. From these observations, we address the question of whether there is a single TATA-binding factor required for the transcription of all genes.

Alleles↗
PubMed1989

Defining the sequence specificity of DNA-binding proteins by selecting binding sites from random-sequence oligonucleotides: analysis of yeast GCN4 protein.

We describe a new method for accurately defining the sequence recognition properties of DNA-binding proteins by selecting high-affinity binding sites from random-sequence DNA. The yeast transcriptional activator protein GCN4 was coupled to a Sepharose column, and binding sites were isolated by passing short, random-sequence oligonucleotides over the column and eluting them with increasing salt concentrations. Of 43 specifically bound oligonucleotides, 40 contained the symmetric sequence TGA(C/G)TCA, whereas the other 3 contained sequences matching six of these seven bases. The extreme preference for this 7-base-pair sequence suggests that each position directly contacts GCN4. The three nucleotide positions on each side of this core heptanucleotide also showed sequence preferences, indicating their effect on GCN4 binding. Interestingly, deviations in the core and a stronger sequence preference in the flanking region were found on one side of the central C . G base pair. Although GCN4 binds as a dimer, this asymmetry supports a model in which interactions on each side of the binding site are not equivalent. The random selection method should prove generally useful for defining the specificities of other DNA-binding proteins and for identifying putative target sequences from genomic DNA.

Base Sequence↗
PubMed1988

Molecular characterization of GCD1, a yeast gene required for general control of amino acid biosynthesis and cell-cycle initiation.

The GCD1 gene product of Saccharomyces cerevisiae has been implicated in the coordination of the cell cycle with the general control of amino acid biosynthesis (M. Wolfner et al., J. Mol. Biol. 96:273-290, 1975). Strains containing the gcd1-1 allele constitutively express the amino acid biosynthetic genes at the induced levels normally found only during conditions of amino acid starvation. In addition, gcd1-1 strains do not grow at high temperatures because under these conditions they are unable to proceed beyond the START step of the cell division cycle. We have cloned and sequenced the GCD1 gene and examined various aspects of cellular metabolism in order to elucidate its role(s) in regulating gene expression and the cell cycle. GCD1 encodes a 1.7 kb RNA whose expression is not regulated as a function of amino acid starvation. Overexpression of this RNA does not affect the regulation of amino acid biosynthetic genes or cell growth. GCD1 is an essential gene because cells containing a gcd1-HIS3 disruption are unable to grow. The essential function of GCD1 may be involved in protein synthesis because a gcd1-1 strain incorporates low levels of 35S-methionine into protein when cells are shifted to the restrictive temperature. GCD1 encodes a protein of 511 amino acids whose predicted sequence does not exhibit significant homology to any other known proteins and appears too large to be a ribosomal protein. We suggest that GCD1 encodes a component of the normal protein synthesis machinery that is involved in the translational regulation of GCN4, a protein that coordinately activates the transcription of amino acid biosynthetic genes. GCD1 may also be part of a sensing mechanism in which cells monitor the protein synthesis capacity prior to initiating a new cell division cycle.

Amino Acid Sequence↗
PubMed1988

Defining the consensus sequences of E.coli promoter elements by random selection.

The consensus sequence of E.coli promoter elements was determined by the method of random selection. A large collection of hybrid molecules was produced in which random-sequence oligonucleotides were cloned in place of a wild-type promoter element, and functional -10 and -35 E.coli promoter elements were obtained by a genetic selection involving the expression of a structural gene. The DNA sequences and relative levels of function for -10 and -35 elements were determined. The consensus sequences determined by this approach are very similar to those determined by comparing DNA sequences of naturally occurring E.coli promoters. However, no strong correlation is observed between similarity to the consensus and relative level of function. The results are considered in terms of E.coli promoter function and of the general applicability of the random selection method.

Base Sequence↗
PubMed1988

Structural and functional characterization of the short acidic transcriptional activation region of yeast GCN4 protein.

Derivatives of the yeast GCN4 transcription factor containing acidic regions of 35 to 40 amino acids fused directly to the DNA-binding domain are fully functional in vivo. High resolution deletion analysis and proteolytic mapping suggest that the activation region is a repeated structure composed of small units acting additively. Acidic character is a feature of the structural motif, possibly a dimer of alpha-helices from two GCN4 monomers, that may be important for interactions with the basic transcriptional machinery.

Amino Acid Sequence↗
PubMed1988

The JUN oncoprotein, a vertebrate transcription factor, activates transcription in yeast.

Transcriptional activation of RNA polymerase II in eukaryotic organisms ranging from yeasts to mammals has many common features such as enhancer elements, TATA elements, and activator proteins that bind specifically to promoter DNA. The JUN oncoprotein, which causes sarcomas in chickens, shows significant homology to the DNA-binding domain of GCN4, a yeast protein that stimulates transcription of the amino acid biosynthetic genes. The GCN4 and JUN proteins bind the same DNA sequences, consensus ATGA(C/G)TCAT, even though the DNA-binding domains are only 45% identical in amino acid sequence. The JUN protein almost certainly represents the oncogenic version of the normal AP-1 transcription factor, suggesting an evolutionary relationship between yeast and vertebrate activator proteins. Here, I demonstrate that JUN efficiently activates transcription in yeast either through its own or a heterologous DNA-binding domain. As is the case for yeast activator proteins, transcriptional stimulation by JUN requires an acidic activation region distinct from the DNA-binding domain. The functional interchangeability between yeast and vertebrate transcription factors strongly suggests a basic similarity in the molecular mechanism of eukaryotic transcriptional activation.

Amino Acid Sequence↗
PubMed1988

Saturation mutagenesis of a yeast his3 "TATA element": genetic evidence for a specific TATA-binding protein.

The yeast his3 promoter region contains two distinct classes of "TATA elements," constitutive (TC) and regulatory (TR), that are defined by their interactions with upstream promoter elements, selectivity of initiation sites, and chromatin structure. TC is localized between positions -83 and -53, and TR is localized between positions -55 and -35, regions in which there are several TATA-like sequences. In this study, we used saturation mutagenesis to examine the structural requirements of the his3 TR element necessary for transcriptional induction. To avoid the complications of redundant elements, the phenotypic analysis was carried out by using a gal-his3 hybrid promoter whose function depends on a short oligonucleotide containing the prospective his3 TR element. In this context, an oligonucleotide containing the sequence TATAAA is sufficient for TR function. However, 17 out of the 18 possible single-base substitutions and 9 out of 10 double mutations of this sequence abolish TR function. This strict sequence requirement for TR function strongly suggests that the TR element is a target site for a sequence-specific DNA-binding protein. Further, as the region encoding TC and promoters of certain other yeast genes do not contain a sequence that is compatible with TR function, we suggest that yeast cells contain multiple proteins with distinct sequence specificities that carry out a related "TATA function" and that yeast promoters can be divided into classes based on their downstream promoter elements.

DNA Mutational Analysis↗
PubMed1988

ATR1, a Saccharomyces cerevisiae gene encoding a transmembrane protein required for aminotriazole resistance.

In Saccharomyces cerevisiae, 3-amino-1,2,4-triazole (aminotriazole) competitively inhibits the activity of imidazoleglycerolphosphate dehydratase, the product of the HIS3 gene. Wild-type strains are able to grow in the presence of 10 mM aminotriazole because they induce the level of imidazoleglycerolphosphate dehydratase. However, strains containing gcn4 mutations are unable to grow in medium containing aminotriazole because they lack the GCN4 transcriptional activator protein necessary for the coordinate induction of HIS3 and other amino acid biosynthetic genes. Here, we isolated a new gene, designated ATR1, which when present in multiple copies per cell allowed gcn4 mutant strains to grow in the presence of aminotriazole. In wild-type strains, multiple copies of ATR1 permitted growth at extremely high concentrations of aminotriazole (80 mM), whereas a chromosomal deletion of ATR1 caused growth inhibition at very low concentrations (5 mM). When radioactive aminotriazole was added exogenously, cells with multiple copies of ATR1 accumulated less aminotriazole than wild-type cells, whereas cells with the atr1 deletion mutation retained more aminotriazole. Unlike the mammalian mdr or yeast PDR genes that confer resistance to many drugs, ATR1 appears to confer resistance only to aminotriazole. Genetic analysis, mRNA mapping, and DNA sequencing revealed that (i) the primary translation product of ATR1 contains 547 amino acids, (ii) ATR1 transcription is induced by aminotriazole, and (iii) the ATR1 promoter region contains a binding site for the GCN4 activator protein. The deduced amino acid sequence suggests that ATR1 protein is very hydrophobic with many membrane-spanning regions, has several potential glycosylation sites, and may contain an ATP-binding site. We suggest that ATR1 encodes a membrane-associated component of the machinery responsible for pumping aminotriazole (and possibly other toxic compounds) out of the cell.

Amino Acid Sequence↗
PubMed1987

Distinguishing between mechanisms of eukaryotic transcriptional activation with bacteriophage T7 RNA polymerase.

To distinguish between mechanisms of eukaryotic transcriptional activation, we tested whether yeast upstream promoter elements can stimulate transcription by a heterologous transcription machinery, bacteriophage T7 RNA polymerase. The gal enhancer-like element recognized by GAL4 protein or the ded1 poly(dA-dT) element was placed upstream of the T7 promoter and his3 structural gene, and T7 RNA polymerase was produced in yeast cells. Under conditions where the gal element would normally be either activating or nonactivating, his3 transcription by T7 RNA polymerase was not stimulated above the level observed in the absence of any upstream element. In contrast, the ded1 poly(dA-dT) element stimulated transcription 7-fold, similar to the enhancement observed on the native ded1 promoter. Activation by the ded1 element thus may involve effects on the chromatin template that facilitate entry of the transcription machinery, whereas activation by the gal element may involve specific contacts between GAL4 and the transcriptional machinery.

DNA-Binding Proteins↗
PubMed1987

The DNA-binding domains of the jun oncoprotein and the yeast GCN4 transcriptional activator protein are functionally homologous.

The jun oncoprotein, which causes sarcomas in chickens, and the DNA-binding domain of yeast GCN4, which coordinately regulates the expression of amino acid biosynthetic genes, show significant homology. In yeast cells deleted for the GCN4 gene, GCN4 function can be conferred by a hybrid protein in which the GCN4 DNA-binding domain is replaced by the homologous region of jun. Moreover, in strains containing various mutations of the GCN4 binding site in the HIS3 promoter, HIS3 expression is affected similarly by the hybrid protein and by GCN4. These results indicate that the jun oncoprotein binds the same DNA sequences as GCN4, and strongly suggest that jun is derived from a normal cellular transcription factor (possibly AP-1, which recognizes similar sequences). This provides direct evidence for the idea that alterations in the machinery for proper gene expression can lead to the oncogenic state.

Avian Sarcoma Viruses↗