scholarly journals The Anatomy of a Hypoxic Operator in Saccharomyces cerevisiae

Genetics ◽  
1998 ◽  
Vol 150 (4) ◽  
pp. 1429-1441 ◽  
Author(s):  
Jutta Deckert ◽  
Ana Maria Rodriguez Torres ◽  
Soo Myung Hwang ◽  
Alexander J Kastaniotis ◽  
Richard S Zitomer
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Binding Sites ◽  
Mutational Analysis ◽  
Consensus Sequence ◽  
Number Of Genes ◽  
Repression Complex ◽  
Single Base Pair ◽  
Sequence Requirements

Abstract Aerobic repression of the hypoxic genes of Saccharomyces cerevisiae is mediated by the DNA-binding protein Rox1 and the Tup1/Ssn6 general repression complex. To determine the DNA sequence requirements for repression, we carried out a mutational analysis of the consensus Rox1-binding site and an analysis of the arrangement of the Rox1 sites into operators in the hypoxic ANB1 gene. We found that single base pair substitutions in the consensus sequence resulted in lower affinities for Rox1, and the decreased affinity of Rox1 for mutant sites correlated with the ability of these sites to repress expression of the hypoxic ANB1 gene. In addition, there was a general but not complete correlation between the strength of repression of a given hypoxic gene and the compliance of the Rox1 sites in that gene to the consensus sequence. An analysis of the ANB1 operators revealed that the two Rox1 sites within an operator acted synergistically in vivo, but that Rox1 did not bind cooperatively in vitro, suggesting the presence of a higher order repression complex in the cell. In addition, the spacing or helical phasing of the Rox1 sites was not important in repression. The differential repression by the two operators of the ANB1 gene was found to be due partly to the location of the operators and partly to the sequences between the two Rox1-binding sites in each. Finally, while Rox1 repression requires the Tup1/Ssn6 general repression complex and this complex has been proposed to require the aminoterminal regions of histones H3 and H4 for full repression of a number of genes, we found that these regions were dispensable for ANB1 repression and the repression of two other hypoxic genes.

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.

A synthetic silencer mediates SIR-dependent functions in Saccharomyces cerevisiae

1991 ◽  
Vol 11 (11) ◽  
pp. 5648-5659
Author(s):  
F J McNally ◽  
J Rine
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Binding Site ◽  
Binding Sites ◽  
Consensus Sequence ◽  
Protein A ◽  
Replication Initiation ◽  
Yeast Saccharomyces Cerevisiae ◽  
Autonomously Replicating Sequence ◽  
Functional Components

Copies of the mating-type genes are present at three loci on chromosome III of the yeast Saccharomyces cerevisiae. The genes at the MAT locus are transcribed, whereas the identical genes at the silent loci, HML and HMR, are not transcribed. Several genes, including the four SIR genes, and two sites, HMR-E and HMR-I, are required for repression of transcription at the HMR locus. Three elements have been implicated in the function of the HMR-E silencer: a binding site for the RAP1 protein, a binding site for the ABF1 protein, and an 11-bp consensus sequence common to nearly all autonomously replicating sequence (ARS) elements (putative origins of DNA replication). RAP1 and ABF1 binding sites of different sequence than those found at HMR-E were joined with an 11-bp ARS consensus sequence to form a synthetic silencer. The synthetic silencer was able to repress transcription of the HMRa1 gene, confirming that binding sites for RAP1 and ABF1 and the 11-bp ARS consensus sequence were the functional components of the silencer in vivo. Mutations in the ABF1 binding site or in the ARS consensus sequence of the synthetic silencer caused nearly complete derepression of transcription at HMR. The ARS consensus sequence mutation also eliminated the ARS activity of the synthetic silencer. These data suggested that replication initiation at the HMR-E silencer was required for establishment of the repressed state at the HMR locus.

scholarly journals Participation of the yeast activator Abf1 in meiosis-specific expression of the HOP1 gene.

1996 ◽  
Vol 16 (6) ◽  
pp. 2777-2786 ◽  
Author(s):  
V Gailus-Durner ◽  
J Xie ◽  
C Chintamaneni ◽  
A K Vershon
Keyword(s):  
Transcriptional Activation ◽  
Mutational Analysis ◽  
Consensus Sequence ◽  
Promoter Sequence ◽  
Regulatory Elements ◽  
Specific Gene ◽  
Specific Expression ◽  
Autonomously Replicating Sequence

The meiosis-specific gene HOP1, which encodes a component of the synaptonemal complex, is controlled through two regulatory elements, UASH and URS1H. Sites similar to URS1H have been identified in the promoter region of virtually every early meiosis-specific gene, as well as in many promoters of nonmeiotic genes, and it has been shown that the proteins that bind to this site function to regulate meiotic and nonmeiotic transcription. Sites similar to the UASH site have been found in a number of meiotic and nonmeiotic genes as well. Since it has been shown that UASH functions as an activator site in vegetative haploid cells, it seemed likely that the factors binding to this site regulate both meiotic and nonmeiotic transcription. We purified the factor binding to the UASH element of the HOP1 promoter. Sequence analysis identified the protein as Abf1 (autonomously replicating sequence-binding factor 1), a multifunctional protein involved in DNA replication, silencing, and transcriptional regulation. We show by mutational analysis of the UASH site, that positions outside of the proposed UASH consensus sequence (TNTGN[A/T]GT) are required for DNA binding in vitro and transcriptional activation in vivo. A new UASH consensus sequence derived from this mutational analysis closely matches a consensus Abf1 binding site. We also show that an Abf1 site from a nonmeiotic gene can replace the function of the UASH site in the HOP1 promoter. Taken together, these results show that Abf1 functions to regulate meiotic gene expression.

scholarly journals PIN*POINT analysis on the endogenous MnSOD promoter: specific demonstration of Sp1 binding in vivo

2003 ◽  
Vol 284 (2) ◽  
pp. C528-C534 ◽  
Author(s):  
Shiuhyang Kuo ◽  
Ann L. Chokas ◽  
Richard J. Rogers ◽  
Harry S. Nick
Keyword(s):  
Binding Sites ◽  
Manganese Superoxide Dismutase ◽  
Consensus Sequence ◽  
Dimethyl Sulfate ◽  
Site Directed Mutagenesis ◽  
Cellular Respiration ◽  
Point Analysis ◽  
Specific Regulation

Manganese superoxide dismutase (MnSOD) is a critical antioxidant enzyme that protects against superoxide anion generated as a consequence of normal cellular respiration, as well as during the inflammatory response. By employing dimethyl sulfate in vivo footprinting, we have previously identified ten basal protein binding sites within the MnSODpromoter. On the basis of consensus sequence comparison and in vitro footprinting data, one would predict that Sp1 might occupy five of these binding sites. To address these findings in the context of the nucleoprotein environment, we first utilized chromatin immunoprecipitation (ChIP) to demonstrate the nuclear association of Sp1 with the MnSOD promoter region. To identify the precise location of Sp1 binding, we have modified the original protein position identification with nuclease tail (PIN*POINT) methodology, providing an approach to establish both the identity and binding occupancy of Sp1 in the context of the endogenous MnSOD promoter. These data, coupled with site-directed mutagenesis, demonstrate the functional importance of two of the Sp1 binding sites in the stimulus-specific regulation of MnSOD gene expression. We feel that the combination of ChIP and PIN*POINT analysis allows unequivocal identification and localization of protein/DNA interactions in vivo, specifically the demonstration of Sp1 with the MnSODpromoter.

scholarly journals The MCP silencer of theDrosophila Abd-Bgene requires both Pleiohomeotic and GAGA factor for the maintenance of repression

Development ◽  
2001 ◽  
Vol 128 (11) ◽  
pp. 2163-2173 ◽  
Author(s):  
Ana Busturia ◽  
Alan Lloyd ◽  
Fernando Bejarano ◽  
Michael Zavortink ◽  
Hua Xin ◽  
...  
Keyword(s):  
Reporter Gene ◽  
Binding Sites ◽  
Mutational Analysis ◽  
Regulatory Elements ◽  
Homeotic Gene ◽  
Gaga Factor ◽  
Polycomb Response Elements ◽  
Silencer Element

Silencing of homeotic gene expression requires the function of cis-regulatory elements known as Polycomb Response Elements (PREs). The MCP silencer element of the Drosophila homeotic gene Abdominal-B has been shown to behave as a PRE and to be required for silencing throughout development. Using deletion analysis and reporter gene assays, we defined a 138 bp sequence within the MCP silencer that is sufficient for silencing of a reporter gene in the imaginal discs. Within the MCP138 fragment, there are four binding sites for the Pleiohomeotic protein (PHO) and two binding sites for the GAGA factor (GAF), encoded by the Trithorax-like gene. PHO and the GAF proteins bind to these sites in vitro. Mutational analysis of PHO and GAF binding sequences indicate that these sites are necessary for silencing in vivo. Moreover, silencing by MCP138 depends on the function of the Trithorax-like gene, and on the function of the PcG genes, including pleiohomeotic. Deletion and mutational analyses show that, individually, either PHO or GAF binding sites retain only weak silencing activity. However, when both PHO and GAF binding sites are present, they achieve strong silencing. We present a model in which robust silencing is achieved by sequential and facilitated binding of PHO and GAF.

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

1990 ◽  
Vol 10 (8) ◽  
pp. 3859-3867
Author(s):  
C R Wobbe ◽  
K Struhl
Keyword(s):  
Dna Sequence ◽  
Consensus Sequence ◽  
Yeast Cells ◽  
Tata Element ◽  
Binding Factor ◽  
Transcription In Vitro ◽  
Sequence Requirements ◽  
Element Function

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.

Identification of Schizosaccharomyces pombe transcription factor PGA4, which binds cooperatively to Saccharomyces cerevisiae GAL4-binding sites

1990 ◽  
Vol 10 (4) ◽  
pp. 1432-1438
Author(s):  
D M Ruden
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transcription Factor ◽  
Schizosaccharomyces Pombe ◽  
Binding Sites ◽  
Transcription Activator ◽  
Integral Number ◽  
Dna Binding Site ◽  
Adh1 Gene

When the DNA-binding site for the Saccharomyces cerevisiae transcription activator GAL4 is placed upstream of the Schizosaccharomyces pombe ADH1 TATA box, transcription of the ADH1 gene is activated in S. pombe in vivo by an endogenous transcription factor. In vitro studies show that this S. pombe protein, PGA4, binds specifically to DNA containing a GAL4 site and that when two GAL4 sites are present, this protein binds cooperatively. Cooperating binding of PGA4 to DNA is favored if the GAL4 sites are separated by an integral number of turns of the DNA helix.

scholarly journals A synthetic silencer mediates SIR-dependent functions in Saccharomyces cerevisiae.

1991 ◽  
Vol 11 (11) ◽  
pp. 5648-5659 ◽  
Author(s):  
F J McNally ◽  
J Rine
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Binding Site ◽  
Binding Sites ◽  
Consensus Sequence ◽  
Protein A ◽  
Replication Initiation ◽  
Yeast Saccharomyces Cerevisiae ◽  
Autonomously Replicating Sequence ◽  
Functional Components

Copies of the mating-type genes are present at three loci on chromosome III of the yeast Saccharomyces cerevisiae. The genes at the MAT locus are transcribed, whereas the identical genes at the silent loci, HML and HMR, are not transcribed. Several genes, including the four SIR genes, and two sites, HMR-E and HMR-I, are required for repression of transcription at the HMR locus. Three elements have been implicated in the function of the HMR-E silencer: a binding site for the RAP1 protein, a binding site for the ABF1 protein, and an 11-bp consensus sequence common to nearly all autonomously replicating sequence (ARS) elements (putative origins of DNA replication). RAP1 and ABF1 binding sites of different sequence than those found at HMR-E were joined with an 11-bp ARS consensus sequence to form a synthetic silencer. The synthetic silencer was able to repress transcription of the HMRa1 gene, confirming that binding sites for RAP1 and ABF1 and the 11-bp ARS consensus sequence were the functional components of the silencer in vivo. Mutations in the ABF1 binding site or in the ARS consensus sequence of the synthetic silencer caused nearly complete derepression of transcription at HMR. The ARS consensus sequence mutation also eliminated the ARS activity of the synthetic silencer. These data suggested that replication initiation at the HMR-E silencer was required for establishment of the repressed state at the HMR locus.

scholarly journals Nucleosome depletion alters the chromatin structure of Saccharomyces cerevisiae centromeres.

1990 ◽  
Vol 10 (11) ◽  
pp. 5721-5727 ◽  
Author(s):  
M J Saunders ◽  
E Yeh ◽  
M Grunstein ◽  
K Bloom
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Chromosome Segregation ◽  
Mutational Analysis ◽  
Base Pairs ◽  
Centromere Function ◽  
Nuclease Digestion ◽  
Dna Elements ◽  
Chromosome Iii ◽  
Sequence Requirements

Saccharomyces cerevisiae centromeric DNA is packaged into a highly nuclease-resistant chromatin core of approximately 200 base pairs of DNA. The structure of the centromere in chromosome III is somewhat larger than a 160-base-pair nucleosomal core and encompasses the conserved centromere DNA elements (CDE I, II, and III). Extensive mutational analysis has revealed the sequence requirements for centromere function. Mutations affecting the segregation properties of centromeres also exhibit altered chromatin structures in vivo. Thus the structure, as delineated by nuclease digestion, correlated with functional centromeres. We have determined the contribution of histone proteins to this unique structural organization. Nucleosome depletion by repression of either histone H2B or H4 rendered the cell incapable of chromosome segregation. Histone repression resulted in increased nuclease sensitivity of centromere DNA, with up to 40% of CEN3 DNA molecules becoming accessible to nucleolytic attack. Nucleosome depletion also resulted in an alteration in the distribution of nuclease cutting sites in the DNA surrounding CEN3. These data provide the first indication that authentic nucleosomal subunits flank the centromere and suggest that nucleosomes may be the central core of the centromere itself.

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