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

1990 ◽  
Vol 10 (6) ◽  
pp. 2832-2839 ◽  
Author(s):  
A S Ponticelli ◽  
K Struhl
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transcriptional Activation ◽  
Transcription Initiation ◽  
Molecular Mechanisms ◽  
Initiation Site ◽  
Activator Proteins ◽  
Nuclear Extracts ◽  
Transcription In Vitro

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.

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

1990 ◽  
Vol 10 (6) ◽  
pp. 2832-2839
Author(s):  
A S Ponticelli ◽  
K Struhl
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transcriptional Activation ◽  
Transcription Initiation ◽  
Molecular Mechanisms ◽  
Initiation Site ◽  
Activator Proteins ◽  
Nuclear Extracts ◽  
Transcription In Vitro

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.

scholarly journals Functional Conservation of the Glutamine-Rich Domains of Yeast Gal11 and Human SRC-1 in the Transactivation of Glucocorticoid Receptor Tau 1 in Saccharomyces cerevisiae

2007 ◽  
Vol 28 (3) ◽  
pp. 913-925 ◽  
Author(s):  
Dae-Hwan Kim ◽  
Gwang Sik Kim ◽  
Chul Ho Yun ◽  
Young Chul Lee
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Glucocorticoid Receptor ◽  
Transcriptional Activation ◽  
Mutational Analysis ◽  
Mediator Complex ◽  
Dependent Manner ◽  
Activator Proteins ◽  
Functional Conservation

ABSTRACT The yeast Gal11 protein, a component of the Mediator complex, is required for the transcriptional activation of many class II genes as a physiological target of various activator proteins in vivo. In this study, we identified the yeast (Saccharomyces cerevisiae) Mediator complex as a novel coactivator of the transcriptional activity of the glucocorticoid receptor (GR) tau 1 (τ1), the major transcriptional activation domain of the GR. GR τ1 directly interacted with the Mediator complex in vivo and in vitro in a Gal11 module-dependent manner, and the Gal11p subunit interacted directly with GR τ1. Specific amino acid residues within the glutamine-rich (Qr) domain of Gal11p (residues 116 to 277) were essential for its interaction with GR τ1 and GR τ1 transactivity in yeast, as demonstrated by mutational analysis of the Gal11 Qr domain, which is highly conserved among human steroid receptor coactivator (SRC) proteins. A Gal11p variant, mini-Gal11p, comprised of the Mediator association and Qr domains of Gal11p or chimeric mini-Gal11p containing the Qr domain of SRC-1 could potentiate the GR τ1 transactivity in a gal11Δ yeast strain. These results suggest that there is functional conservation between Qr domains of yeast Gal11p and mammalian SRC proteins as direct targets of activator proteins in yeast.

scholarly journals Positive and Negative Autoregulation ofREB1 Transcription in Saccharomyces cerevisiae

1998 ◽  
Vol 18 (7) ◽  
pp. 4368-4376 ◽  
Author(s):  
Kevin L.-C. Wang ◽  
Jonathan R. Warner
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Binding Sites ◽  
Transcription Initiation ◽  
Transcriptional Control ◽  
Initiation Site ◽  
Transcription Unit ◽  
Pol I ◽  
Activation Of Transcription

ABSTRACT Reb1p is a DNA binding protein of Saccharomyces cerevisiae that has been implicated in the activation of transcription by polymerase (Pol) II, in the termination of transcription by Pol I, and in the organization of nucleosomes. Studies of the transcriptional control of the REB1 gene have led us to identify three Reb1p binding sites in the 5′ region of the its gene, termed A, B, and C, at positions −110, −80, and +30 with respect to transcription initiation. In vitro, Reb1p binds to the three sites with the relative affinity of A ≥ C > B. Kinetic parameters suggest that when both A and C sites are present on the same DNA molecule, the C site may recruit Reb1p for the A site. In vivo the A and B sites each contribute to the transcription activity ofREB1 in roughly additive fashion. Mutation of both A and B sites abolishes transcription. On the other hand, the C site is a negative element, reducing transcription by 40%. In cells overexpressing Reb1p, the C site reduces transcription by more than 80%. This effect can be transposed to another transcription unit, demonstrating that the effect of Reb1p binding at the C site does not depend on interaction with upstream Reb1p molecules. Relocation of the C site to a position 105 bp downstream of the transcription initiation site abolishes its effect, suggesting that it does not act as a conventional attenuator of transcription. We conclude that binding of Reb1p at the C site hinders formation of the initiation complex. This arrangement of Reb1p binding sites provides a positive and negative mechanism to autoregulate the expression of REB1. Such an arrangement could serve to dampen the inevitable fluctuation in Rep1p levels caused by the intermittent presence of its mRNA within an individual cell.

Interspersion of an unusual GCN4 activation site with a complex transcriptional repression site in Ty2 elements of Saccharomyces cerevisiae

1993 ◽  
Vol 13 (4) ◽  
pp. 2091-2103
Author(s):  
S Türkel ◽  
P J Farabaugh
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transcriptional Activation ◽  
Binding Sites ◽  
Transcriptional Repression ◽  
Physiological Control ◽  
Reading Frame ◽  
Negative Region ◽  
Activation Site

Transcription of the Ty2-917 retrotransposon of Saccharomyces cerevisiae is modulated by a complex set of positive and negative elements, including a negative region located within the first open reading frame, TYA2. The negative region includes three downstream repression sites (DRSI, DRSII, and DRSIII). In addition, the negative region includes at least two downstream activation sites (DASs). This paper concerns the characterization of DASI. A 36-bp DASI oligonucleotide acts as an autonomous transcriptional activation site and includes two sequence elements which are both required for activation. We show that these sites bind in vitro the transcriptional activation protein GCN4 and that their activity in vivo responds to the level of GCN4 in the cell. We have termed the two sites GCN4 binding sites (GBS1 and GBS2). GBS1 is a high-affinity GCN4 binding site (dissociation constant, approximately 25 nM at 30 degrees C), binding GCN4 with about the affinity of a consensus UASGCN4, this though GBS1 includes two differences from the right half of the palindromic consensus site. GBS2 is more diverged from the consensus and binds GCN4 with about 20-fold-lower affinity. Nucleotides 13 to 36 of DASI overlap DRSII. Since DRSII is a transcriptional repression site, we tested whether DASI includes repression elements. We identify two sites flanking GBS2, both of which repress transcription activated by the consensus GCN4-specific upstream activation site (UASGCN4). One of these is repeated in the 12 bp immediately adjacent to DASI. Thus, in a 48-bp region of Ty2-917 are interspersed two positive and three negative transcriptional regulators. The net effect of the region must depend on the interaction of the proteins bound at these sites, which may include their competing for binding sites, and on the physiological control of the activity of these proteins.

Identification of promoter elements necessary for transcriptional regulation of a human histone H4 gene in vitro

1985 ◽  
Vol 5 (2) ◽  
pp. 380-389
Author(s):  
S M Hanly ◽  
G C Bleecker ◽  
N Heintz
Keyword(s):  
Transcription Factor ◽  
Transcription Initiation ◽  
Initiation Site ◽  
S Phase ◽  
Histone H4 ◽  
Promoter Elements ◽  
Nuclear Extracts ◽  
Distal Promoter ◽  
Histone H4 Gene

We have examined the nucleotide sequences necessary for transcription of a human histone H4 gene in vitro. Maximal transcription of the H4 promoter requires, in addition to the TATA box and cap site, promoter elements between 70 and 110 nucleotides upstream from the transcription initiation site. These distal promoter elements are recognized preferentially in extracts from synchronized S-phase HeLa cells. The inability of non-S-phase nuclear extracts to recognize the H4 upstream sequences reflects a specific lack of a transcription factor which interacts with those sequences. These results indicate that the cell cycle regulation of human histone gene expression involves both a specific transcription factor and distal transcription signals in the H4 promoter.

scholarly journals Molecular Mechanisms Underlying the Positive Stringent Response of the Bacillus subtilis ilv-leu Operon, Involved in the Biosynthesis of Branched-Chain Amino Acids

2008 ◽  
Vol 190 (18) ◽  
pp. 6134-6147 ◽  
Author(s):  
Shigeo Tojo ◽  
Takenori Satomura ◽  
Kanako Kumamoto ◽  
Kazutake Hirooka ◽  
Yasutaro Fujita
Keyword(s):  
Amino Acids ◽  
Bacillus Subtilis ◽  
Transcription Initiation ◽  
Molecular Mechanisms ◽  
Stringent Response ◽  
Branched Chain Amino Acids ◽  
Base Substitution ◽  
Branched Chain

ABSTRACT Branched-chain amino acids are the most abundant amino acids in proteins. The Bacillus subtilis ilv-leu operon is involved in the biosynthesis of branched-chain amino acids. This operon exhibits a RelA-dependent positive stringent response to amino acid starvation. We investigated this positive stringent response upon lysine starvation as well as decoyinine treatment. Deletion analysis involving various lacZ fusions revealed two molecular mechanisms underlying the positive stringent response of ilv-leu, i.e., CodY-dependent and -independent mechanisms. The former is most likely triggered by the decrease in the in vivo concentration of GTP upon lysine starvation, GTP being a corepressor of the CodY protein. So, the GTP decrease derepressed ilv-leu expression through detachment of the CodY protein from its cis elements upstream of the ilv-leu promoter. By means of base substitution and in vitro transcription analyses, the latter (CodY-independent) mechanism was found to comprise the modulation of the transcription initiation frequency, which likely depends on fluctuation of the in vivo RNA polymerase substrate concentrations after stringent treatment, and to involve at least the base species of adenine at the 5′ end of the ilv-leu transcript. As discussed, this mechanism is presumably distinct from that for B. subtilis rrn operons, which involves changes in the in vivo concentration of the initiating GTP.

scholarly journals Sequences that regulate the divergent GAL1-GAL10 promoter in Saccharomyces cerevisiae.

1984 ◽  
Vol 4 (8) ◽  
pp. 1440-1448 ◽  
Author(s):  
M Johnston ◽  
R W Davis
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Nucleotide Sequence ◽  
Base Pair ◽  
Transcription Initiation ◽  
Base Pairs ◽  
Regulation Of Expression ◽  
Notable Feature ◽  
His3 Gene

The GAL1 and GAL10 genes of Saccharomyces cerevisiae are divergently transcribed, with 606 base pairs of DNA separating their transcription initiation sites. These two genes are stringently coregulated: their expression is induced ca. 1,000-fold in cells growing on galactose and is repressed by growth on glucose. The nucleotide sequence of the region of DNA between these genes and the precise sites of transcription initiation are presented here. The most notable feature of the nucleotide sequence of this region is a 108-base-pair guanine-plus-cytosine-rich stretch of DNA located approximately in the middle of the region between GAL1 and GAL10. Analysis of the effects of mutations that alter the region between these two genes, constructed in vitro or selected in vivo, suggest that these guanine-plus-cytosine-rich sequences are required for the expression of both genes. The region of DNA between GAL1 and GAL10 is sufficient for regulation of expression of these genes: fusion of the region to the yeast HIS3 gene places HIS3 under GAL control.

scholarly journals Interactions of the Antizyme AtoC with Regulatory Elements of the Escherichia coli atoDAEB Operon

2007 ◽  
Vol 189 (17) ◽  
pp. 6324-6332 ◽  
Author(s):  
Meropi K. Matta ◽  
Efthimia E. Lioliou ◽  
Cynthia H. Panagiotidis ◽  
Dimitrios A. Kyriakidis ◽  
Christos A. Panagiotidis
Keyword(s):  
Escherichia Coli ◽  
Transcription Initiation ◽  
Response Regulator ◽  
Regulatory Elements ◽  
Initiation Site ◽  
Integration Host Factor ◽  
Chemical Mutagenesis ◽  
Dual Function

ABSTRACT AtoC has a dual function as both an antizyme, the posttranslational inhibitor of polyamine biosynthetic enzymes, and the transcriptional regulator of genes involved in short-chain fatty acid catabolism (the atoDAEB operon). We have previously shown that AtoC is the response regulator of the AtoS-AtoC two-component signal transduction system that activates atoDAEB when Escherichia coli is exposed to acetoacetate. Here, we show that the same cis elements control both promoter inducibility and AtoC binding. Chromatin immunoprecipitation experiments confirmed the acetoacetate-inducible binding of AtoC to the predicted DNA region in vivo. DNase I protection footprinting analysis revealed that AtoC binds two 20-bp stretches, constituting an inverted palindrome, that are located at −146 to −107 relative to the transcription initiation site. Analyses of promoter mutants obtained by in vitro chemical mutagenesis of the atoDAEB promoter verified both the importance of AtoC binding for the inducibility of the promoter by acetoacetate and the σ54 dependence of atoDAEB expression. The integration host factor was also identified as a critical component of the AtoC-mediated induction of atoDAEB.

scholarly journals The DRIP Complex and SRC-1/p160 Coactivators Share Similar Nuclear Receptor Binding Determinants but Constitute Functionally Distinct Complexes

2000 ◽  
Vol 20 (8) ◽  
pp. 2718-2726 ◽  
Author(s):  
Christophe Rachez ◽  
Matthew Gamble ◽  
Chao-Pei Betty Chang ◽  
G. Brandon Atkins ◽  
Mitchell A. Lazar ◽  
...  
Keyword(s):  
Nuclear Receptors ◽  
Nuclear Receptor ◽  
Receptor Binding ◽  
Transcriptional Activation ◽  
Thyroid Hormone Receptor ◽  
Nuclear Receptor Coactivators ◽  
Nuclear Extracts ◽  
Sequence Requirements

ABSTRACT Transcriptional activation requires both access to DNA assembled as chromatin and functional contact with components of the basal transcription machinery. Using the hormone-bound vitamin D3receptor (VDR) ligand binding domain (LBD) as an affinity matrix, we previously identified a novel multisubunit coactivator complex, DRIP (VDR-interacting proteins), required for transcriptional activation by nuclear receptors and several other transcription factors. In this report, we characterize the nuclear receptor binding features of DRIP205, a key subunit of the DRIP complex, that interacts directly with VDR and thyroid hormone receptor in response to ligand and anchors the other DRIP subunits to the nuclear receptor LBD. In common with other nuclear receptor coactivators, DRIP205 interaction occurs through one of two LXXLL motifs and requires the receptor's AF-2 subdomain. Although the second motif of DRIP205 is required only for VDR binding in vitro, both motifs are used in the context of an retinoid X receptor-VDR heterodimer on DNA and in transactivation in vivo. We demonstrate that both endogenous p160 coactivators and DRIP complexes bind to the VDR LBD from nuclear extracts through similar sequence requirements, but they do so as distinct complexes. Moreover, in contrast to the p160 family of coactivators, the DRIP complex is devoid of any histone acetyltransferase activity. The results demonstrate that different coactivator complexes with distinct functions bind to the same transactivation region of nuclear receptors, suggesting that they are both required for transcription activation by nuclear receptors.

scholarly journals Yeast Coactivator MBF1 Mediates GCN4-Dependent Transcriptional Activation

1998 ◽  
Vol 18 (9) ◽  
pp. 4971-4976 ◽  
Author(s):  
Ken-ichi Takemaru ◽  
Satoshi Harashima ◽  
Hitoshi Ueda ◽  
Susumu Hirose
Keyword(s):  
Gene Expression ◽  
Saccharomyces Cerevisiae ◽  
Dna Binding ◽  
Nuclear Receptor ◽  
Transcriptional Activation ◽  
Crucial Role ◽  
Tata Binding Protein ◽  
Binding Region

ABSTRACT Transcriptional coactivators play a crucial role in gene expression by communicating between regulatory factors and the basal transcription machinery. The coactivator multiprotein bridging factor 1 (MBF1) was originally identified as a bridging molecule that connects theDrosophila nuclear receptor FTZ-F1 and TATA-binding protein (TBP). The MBF1 sequence is highly conserved across species fromSaccharomyces cerevisiae to human. Here we provide evidence acquired in vitro and in vivo that yeast MBF1 mediates GCN4-dependent transcriptional activation by bridging the DNA-binding region of GCN4 and TBP. These findings indicate that the coactivator MBF1 functions by recruiting TBP to promoters where DNA-binding regulators are bound.

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