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.

scholarly journals 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.

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

1990 ◽  
Vol 10 (8) ◽  
pp. 4256-4265 ◽  
Author(s):  
C J Brandl ◽  
K Struhl
Keyword(s):  
Binding Site ◽  
Transcriptional Activation ◽  
Nucleosome Positioning ◽  
Alternative Mechanism ◽  
Tata Element ◽  
Binding Factor ◽  
Positioning Analysis ◽  
Binding Sequence

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.

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

1990 ◽  
Vol 10 (8) ◽  
pp. 4256-4265
Author(s):  
C J Brandl ◽  
K Struhl
Keyword(s):  
Binding Site ◽  
Transcriptional Activation ◽  
Nucleosome Positioning ◽  
Alternative Mechanism ◽  
Tata Element ◽  
Binding Factor ◽  
Positioning Analysis ◽  
Binding Sequence

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.

An essential yeast protein, CBF5p, binds in vitro to centromeres and microtubules

1993 ◽  
Vol 13 (8) ◽  
pp. 4884-4893
Author(s):  
W Jiang ◽  
K Middleton ◽  
H J Yoon ◽  
C Fouquet ◽  
J Carbon
Keyword(s):  
Topoisomerase Ii ◽  
Yeast Cells ◽  
Affinity Column ◽  
Bacterial Cells ◽  
Binding Domains ◽  
C Terminus ◽  
Binding Factor ◽  
Associated Proteins

Yeast centromere DNA (CEN) affinity column chromatography has been used to purify several putative centromere and kinetochore proteins from yeast chromatin extracts. The single yeast gene (CBF5) specifying one of the major low-affinity centromere-binding proteins (p64'/CBF5p) has been cloned and shown to be essential for viability of Saccharomyces cerevisiae. CBF5 specifies a 55-kDa highly charged protein that contains a repeating KKD/E sequence domain near the C terminus, similar to known microtubule-binding domains in microtubule-associated proteins 1A and 1B, CBF5p, obtained by overexpression in bacterial cells, binds microtubules in vitro, whereas C-terminal deleted proteins lacking the (KKD/E)n domain do not. Dividing yeast cells containing a C-terminal truncated CBF5 gene, producing CBF5p containing only three copies of the KKD/E repeat, delay with replicated genomes at the G2/M phase of the cell cycle, while depletion of CBF5p arrests most cells in G1/S. Overproduction of CBF5p in S. cerevisiae complements a temperature sensitivity mutation in the gene (CBF2) specifying the 110-kDa subunit of the high-affinity CEN DNA-binding factor CBF3, suggesting in vivo interaction of CBF5p and CBF3. A second low-affinity centromere-binding factor has been identified as topoisomerase II.

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.

DNA sequence requirement of a TATA element-binding protein from Arabidopsis for transcription in vitro

1993 ◽  
Vol 23 (5) ◽  
pp. 995-1003 ◽  
Author(s):  
Fujio Mukumoto ◽  
Susumu Hirose ◽  
Hidemasa Imaseki ◽  
Ken-ichi Yamazaki
Keyword(s):  
Dna Sequence ◽  
Binding Protein ◽  
Tata Element ◽  
Element Binding Protein ◽  
Transcription In Vitro

scholarly journals Analysis of Core Promoter Sequences Located Downstream from the TATA Element in the hsp70 Promoter from Drosophila melanogaster

2001 ◽  
Vol 21 (5) ◽  
pp. 1593-1602 ◽  
Author(s):  
Chwen-Huey Wu ◽  
Lakshmi Madabusi ◽  
Hokuto Nishioka ◽  
Peter Emanuel ◽  
Michael Sypes ◽  
...  
Keyword(s):  
Core Promoter ◽  
P Element ◽  
Multiple Sequence ◽  
Promoter Sequences ◽  
Tata Element ◽  
Sequence Elements ◽  
Cross Links ◽  
Transcription In Vitro

ABSTRACT TFIID recognizes multiple sequence elements in thehsp70 promoter of Drosophila. Here, we investigate the function of sequences downstream from the TATA element. A mutation in the initiator was identified that caused an eightfold reduction in binding of TFIID and a fourfold reduction in transcription in vitro. Another mutation in the +24 to +29 region was somewhat less inhibitory, but a mutation in the +14 to +19 region had essentially no effect. The normal promoter and the mutants in the initiator and the +24 to +29 region were transformed into flies by P element-mediated transformation. The initiator mutation reduced expression an average of twofold in adult flies, whereas the mutation in the +24 to +29 region had essentially no effect. In contrast, a promoter combining the two mutations was expressed an average of sixfold less than the wild type. The results suggest that the initiator and the +24 to +29 region could serve overlapping functions in vivo. Protein-DNA cross-linking was used to identify which subunits of TFIID contact the +24 to +29 region and the initiator. No specific subunits were found to cross-link to the +24 to +29 region. In contrast, the initiator cross-linked exclusively to dTAF230. Remarkably, dTAF230 cross-links approximately 10 times more efficiently to the nontranscribed strand than to the transcribed strand at the initiator.

scholarly journals Genomewide Identification of Sko1 Target Promoters Reveals a Regulatory Network That Operates in Response to Osmotic Stress in Saccharomyces cerevisiae

2005 ◽  
Vol 4 (8) ◽  
pp. 1343-1352 ◽  
Author(s):  
Markus Proft ◽  
Francis D. Gibbons ◽  
Matthew Copeland ◽  
Frederick P. Roth ◽  
Kevin Struhl
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transcription Factors ◽  
Osmotic Stress ◽  
Consensus Sequence ◽  
Signal Transduction Pathway ◽  
Mitogen Activated Protein Kinase ◽  
Yeast Cells ◽  
Target Promoters

ABSTRACT In Saccharomyces cerevisiae, the ATF/CREB transcription factor Sko1 (Acr1) regulates the expression of genes induced by osmotic stress under the control of the high osmolarity glycerol (HOG) mitogen-activated protein kinase pathway. By combining chromatin immunoprecipitation and microarrays containing essentially all intergenic regions, we estimate that yeast cells contain approximately 40 Sko1 target promoters in vivo; 20 Sko1 target promoters were validated by direct analysis of individual loci. The ATF/CREB consensus sequence is not statistically overrepresented in confirmed Sko1 target promoters, although some sites are evolutionarily conserved among related yeast species, suggesting that they are functionally important in vivo. These observations suggest that Sko1 association in vivo is affected by factors beyond the protein-DNA interaction defined in vitro. Sko1 binds a number of promoters for genes directly involved in defense functions that relieve osmotic stress. In addition, Sko1 binds to the promoters of genes encoding transcription factors, including Msn2, Mot3, Rox1, Mga1, and Gat2. Stress-induced expression of MSN2, MOT3, and MGA1 is diminished in sko1 mutant cells, while transcriptional regulation of ROX1 seems to be unaffected. Lastly, Sko1 targets PTP3, which encodes a phosphatase that negatively regulates Hog1 kinase activity, and Sko1 is required for osmotic induction of PTP3 expression. Taken together our results suggest that Sko1 operates a transcriptional network upon osmotic stress, which involves other specific transcription factors and a phosphatase that regulates the key component of the signal transduction pathway.

scholarly journals Half-Site DNA Sequence and Spacing Length Contributions to PrrA Binding to PrrA Site 2 of RSP3361 in Rhodobacter sphaeroides 2.4.1

2009 ◽  
Vol 191 (13) ◽  
pp. 4353-4364 ◽  
Author(s):  
Jesus M. Eraso ◽  
Samuel Kaplan
Keyword(s):  
Dna Binding ◽  
Dna Sequence ◽  
Rhodobacter Sphaeroides ◽  
Dna Sequences ◽  
Mutational Analysis ◽  
Consensus Sequence ◽  
Binding Affinities ◽  
Transcriptional Fusions

ABSTRACT The consensus DNA binding sequence for PrrA, a global regulator in Rhodobacter sphaeroides 2.4.1, is poorly defined. We have performed mutational analysis of PrrA site 2, of the RSP3361 gene, to which PrrA binds in vitro (J. M. Eraso and S. Kaplan, J. Bacteriol. 191:4341-4352, 2009), to further define the consensus sequence for DNA binding. Two half-sites of equal length, containing 6 nucleotides each, were required for PrrA binding to this DNA sequence. Systematic nucleotide substitutions in both inverted half-sites led to a decrease in binding affinity of phosphorylated PrrA in vitro, the level of which was dependent on the substitution. The reduced binding affinities were confirmed by competition experiments and led to proportional decreases in the expression of lacZ transcriptional fusions to the RSP3361 gene in vivo. The 5-nucleotide spacer region between the half-sites was found to be optimal for PrrA binding to the wild-type half-sites, as shown by decreased PrrA DNA binding affinities to synthetic DNA sequences without spacer regions or with spacer regions ranging from 1 to 10 nucleotides. The synthetic spacer region alleles also showed decreased gene expression in vivo when analyzed using lacZ transcriptional fusions. We have studied three additional DNA sequences to which PrrA binds in vitro. They are located in the regulatory regions of genes positively regulated by PrrA and contain spacer regions with 5 or 8 nucleotides. We demonstrate that PrrA can bind in vitro to DNA sequences with different lengths in the spacer regions between the half-sites.

scholarly journals An essential yeast protein, CBF5p, binds in vitro to centromeres and microtubules.

1993 ◽  
Vol 13 (8) ◽  
pp. 4884-4893 ◽  
Author(s):  
W Jiang ◽  
K Middleton ◽  
H J Yoon ◽  
C Fouquet ◽  
J Carbon
Keyword(s):  
Topoisomerase Ii ◽  
Yeast Cells ◽  
Affinity Column ◽  
Bacterial Cells ◽  
Binding Domains ◽  
C Terminus ◽  
Binding Factor ◽  
Associated Proteins

Yeast centromere DNA (CEN) affinity column chromatography has been used to purify several putative centromere and kinetochore proteins from yeast chromatin extracts. The single yeast gene (CBF5) specifying one of the major low-affinity centromere-binding proteins (p64'/CBF5p) has been cloned and shown to be essential for viability of Saccharomyces cerevisiae. CBF5 specifies a 55-kDa highly charged protein that contains a repeating KKD/E sequence domain near the C terminus, similar to known microtubule-binding domains in microtubule-associated proteins 1A and 1B, CBF5p, obtained by overexpression in bacterial cells, binds microtubules in vitro, whereas C-terminal deleted proteins lacking the (KKD/E)n domain do not. Dividing yeast cells containing a C-terminal truncated CBF5 gene, producing CBF5p containing only three copies of the KKD/E repeat, delay with replicated genomes at the G2/M phase of the cell cycle, while depletion of CBF5p arrests most cells in G1/S. Overproduction of CBF5p in S. cerevisiae complements a temperature sensitivity mutation in the gene (CBF2) specifying the 110-kDa subunit of the high-affinity CEN DNA-binding factor CBF3, suggesting in vivo interaction of CBF5p and CBF3. A second low-affinity centromere-binding factor has been identified as topoisomerase II.

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