scholarly journals Identification of functional regions in the yeast transcriptional activator ADR1.

1988 ◽  
Vol 8 (5) ◽  
pp. 2125-2131 ◽  
Author(s):  
L T Bemis ◽  
C L Denis
Keyword(s):  
Amino Acids ◽  
Dna Binding ◽  
Transcriptional Activation ◽  
Glucose Repression ◽  
Transcriptional Activator ◽  
Dna Binding Protein ◽  
Functional Regions ◽  
Zinc Finger Motifs ◽  
Repressor Molecule ◽  
Carboxy Terminal

The transcriptional activator ADR1 from Saccharomyces cerevisiae is a postulated DNA-binding protein that controls the expression of the glucose-repressible alcohol dehydrogenase (ADH2). Carboxy-terminal deletions of the ADR1 protein (1,323 amino acids in length) were used to localize its functional regions. The transcriptional activation region was localized to the N-terminal 220 amino acids of ADR1 containing two DNA-binding zinc finger motifs. In addition to the N terminus, a large part of the ADR1 sequence was shown to be essential for complete activation of ADH2. Deletion of the putative phosphorylation region, defined by ADR1c mutations that overcome glucose repression, did not render ADH2 expression insensitive to glucose repression. Instead, this region (amino acids 220 through 253) was found to be required by ADR1 to bypass glucose repression. These results suggest that ADR1c mutations enhance ADR1 function, rather than block an interaction of the putative phosphorylation region with a repressor molecule. Furthermore, the protein kinase CCR1 was shown to affect ADH2 expression when the putative phosphorylation region was removed, indicating that CCR1 does not act solely through this region. A functional ADR1 gene was also found to be necessary for growth on glycerol-containing medium. The N-terminal 506 amino acids of ADR1 were required for this newly identified function, indicating that ADH2 activation and glycerol growth are controlled by separate regions of ADR1.

Identification of functional regions in the yeast transcriptional activator ADR1

1988 ◽  
Vol 8 (5) ◽  
pp. 2125-2131
Author(s):  
L T Bemis ◽  
C L Denis
Keyword(s):  
Amino Acids ◽  
Dna Binding ◽  
Transcriptional Activation ◽  
Glucose Repression ◽  
Transcriptional Activator ◽  
Dna Binding Protein ◽  
Functional Regions ◽  
Zinc Finger Motifs ◽  
Repressor Molecule ◽  
Carboxy Terminal

The transcriptional activator ADR1 from Saccharomyces cerevisiae is a postulated DNA-binding protein that controls the expression of the glucose-repressible alcohol dehydrogenase (ADH2). Carboxy-terminal deletions of the ADR1 protein (1,323 amino acids in length) were used to localize its functional regions. The transcriptional activation region was localized to the N-terminal 220 amino acids of ADR1 containing two DNA-binding zinc finger motifs. In addition to the N terminus, a large part of the ADR1 sequence was shown to be essential for complete activation of ADH2. Deletion of the putative phosphorylation region, defined by ADR1c mutations that overcome glucose repression, did not render ADH2 expression insensitive to glucose repression. Instead, this region (amino acids 220 through 253) was found to be required by ADR1 to bypass glucose repression. These results suggest that ADR1c mutations enhance ADR1 function, rather than block an interaction of the putative phosphorylation region with a repressor molecule. Furthermore, the protein kinase CCR1 was shown to affect ADH2 expression when the putative phosphorylation region was removed, indicating that CCR1 does not act solely through this region. A functional ADR1 gene was also found to be necessary for growth on glycerol-containing medium. The N-terminal 506 amino acids of ADR1 were required for this newly identified function, indicating that ADH2 activation and glycerol growth are controlled by separate regions of ADR1.

AND-1, a natural chimeric DNA-binding protein, combines an HMG-box with regulatory WD-repeats

1997 ◽  
Vol 110 (9) ◽  
pp. 1051-1062 ◽  
Author(s):  
A. Kohler ◽  
M.S. Schmidt-Zachmann ◽  
W.W. Franke
Keyword(s):  
Amino Acids ◽  
Dna Binding ◽  
Binding Protein ◽  
Dna Binding Protein ◽  
Sequence Motifs ◽  
Mobility Shift ◽  
Hmg Box ◽  
Amino Terminal ◽  
Wd Repeats ◽  
Carboxy Terminal

Using a specific monoclonal antibody (mAb AND-1/23-5-14) we have identified, cDNA-cloned and characterized a novel DNA-binding protein of the clawed toad, Xenopus laevis, that is accumulated in the nucleoplasm of oocytes and various other cells. This protein comprises 1,127 amino acids, with a total molecular mass of 125 kDa and a pI of 5.27. It is encoded by a mRNA of approximately 4 kb and contains, in addition to clusters of acidic amino acids, two hallmark motifs: the amino-terminal part harbours seven consecutive ‘WD-repeats’, which are sequence motifs of about 40 amino acids that are characteristic of a large group of regulatory proteins involved in diverse cellular functions, while the carboxy terminal portion possesses a 63-amino-acid-long ‘HMG-box’, which is typical of a family of DNA-binding proteins involved in regulation of chromatin assembly, transcription and replication. The DNA-binding capability of the protein was demonstrated by DNA affinity chromatography and electrophoretic mobility shift assays using four-way junction DNA. Protein AND-1 (acidic nucleoplasmic DNA-binding protein) appears as an oligomer, probably a homodimer, and has been localized throughout the entire interchromatinic space of the interphase nucleoplasm, whereas during mitosis it is transiently dispersed over the cytoplasm. We also identified a closely related, perhaps orthologous protein in mammals. The unique features of protein AND-1, which is a ‘natural chimera’ combining properties of the WD-repeat and the HMG-box families of proteins, are discussed in relation to its possible nuclear functions.

scholarly journals Interdigitated residues within a small region of VP16 interact with Oct-1, HCF, and DNA.

1997 ◽  
Vol 17 (7) ◽  
pp. 3937-3946 ◽  
Author(s):  
J S Lai ◽  
W Herr
Keyword(s):  
Amino Acids ◽  
Dna Binding ◽  
Transcriptional Activation ◽  
Small Region ◽  
Transcriptional Activation Domain ◽  
Amino Acid Region ◽  
Transcriptional Regulatory ◽  
Mat Alpha2 ◽  
Simplex Virus ◽  
Carboxy Terminal

Upon infection, the herpes simplex virus (HSV) activator of immediate-early (IE) gene transcription VP16 forms a multiprotein-DNA complex with two cellular proteins, Oct-1 and HCF. First, VP16 associates with HCF independently of DNA, and this association stimulates subsequent association with Oct-1 on the DNA target of VP16 activation, the TAATGARAT motif found in HSV IE promoters. We have analyzed the involvement of VP16 residues lying near the carboxy-terminal transcriptional activation domain of VP16 in associating with HCF, Oct-1, and DNA. To assay VP16 association with HCF, we developed an electrophoretic mobility retardation assay in which HCF is used to retard the mobility of a hybrid VP16-GAL4 DNA-binding domain fusion protein bound to a GAL4 DNA-binding site. Analysis of an extensive set of individual and combined alanine substitutions over a 61-amino-acid region of VP16 shows that, even within a region as small as 13 amino acids, there are separate residues involved in association with either HCF, DNA, or Oct-1 bound to DNA; indeed, of two immediately adjacent amino acids in VP16, one is important for DNA binding and the other is important for HCF binding. These results suggest that a small region in VP16 is important for linking in close juxtaposition the four components of the VP16-induced complex and support the hypothesis that the structure of the Oct-1-VP16 interaction in this complex is similar to that formed by the yeast transcriptional regulatory proteins MATa1 and MAT alpha2. We propose that HCF stabilizes this Oct-1-VP16 interaction.

scholarly journals The E-box DNA binding protein Sgc1p suppresses thegcr2mutation, which is involved in transcriptional activation of glycolytic genes inSaccharomyces cerevisiae

FEBS Letters ◽  
1999 ◽  
Vol 463 (3) ◽  
pp. 307-311 ◽  
Author(s):  
Takashi Sato ◽  
M.Cecilia Lopez ◽  
Shigemi Sugioka ◽  
Yoshifumi Jigami ◽  
Henry V. Baker ◽  
...  
Keyword(s):  
Dna Binding ◽  
Transcriptional Activation ◽  
Binding Protein ◽  
Dna Binding Protein ◽  
E Box

scholarly journals Recruitment of TATA-Binding Protein–TAFI Complex SL1 to the Human Ribosomal DNA Promoter Is Mediated by the Carboxy-Terminal Activation Domain of Upstream Binding Factor (UBF) and Is Regulated by UBF Phosphorylation

1999 ◽  
Vol 19 (4) ◽  
pp. 2872-2879 ◽  
Author(s):  
JoAnn C. Tuan ◽  
Weiguo Zhai ◽  
Lucio Comai
Keyword(s):  
Dna Binding ◽  
Ribosomal Dna ◽  
Transcriptional Activation ◽  
Direct Contact ◽  
Binding Protein ◽  
Tata Binding Protein ◽  
Rrna Synthesis ◽  
Binding Factor ◽  
Upstream Binding Factor ◽  
Carboxy Terminal

ABSTRACT Human rRNA synthesis by RNA polymerase I requires at least two auxiliary factors, upstream binding factor (UBF) and SL1. UBF is a DNA binding protein with multiple HMG domains that binds directly to the CORE and UCE elements of the ribosomal DNA promoter. The carboxy-terminal region of UBF is necessary for transcription activation and has been shown to be extensively phosphorylated. SL1, which consists of TATA-binding protein (TBP) and three associated factors (TAFIs), does not have any sequence-specific DNA binding activity, and its recruitment to the promoter is mediated by specific protein interactions with UBF. Once on the promoter, the SL1 complex makes direct contact with the DNA promoter and directs promoter-specific initiation of transcription. To investigate the mechanism of UBF-dependent transcriptional activation, we first performed protein-protein interaction assays between SL1 and a series of UBF deletion mutants. This analysis indicated that the carboxy-terminal domain of UBF, which is necessary for transcriptional activation, makes direct contact with the TBP-TAFI complex SL1. Since this region of UBF can be phosphorylated, we then tested whether this modification plays a functional role in the interaction with SL1. Alkaline phosphatase treatment of UBF completely abolished the ability of UBF to interact with SL1; moreover, incubation of the dephosphorylated UBF with nuclear extracts from exponentially growing cells was able to restore the UBF-SL1 interaction. In addition, DNase I footprinting analysis and in vitro-reconstituted transcription assays with phosphatase-treated UBF provided further evidence that UBF phosphorylation plays a critical role in the regulation of the recruitment of SL1 to the ribosomal DNA promoter and stimulation of UBF-dependent transcription.

scholarly journals Dissection of a carboxy-terminal region of the yeast regulatory protein RAP1 with effects on both transcriptional activation and silencing

1992 ◽  
Vol 12 (3) ◽  
pp. 1209-1217
Author(s):  
C F Hardy ◽  
D Balderes ◽  
D Shore
Keyword(s):  
Dna Binding ◽  
Binding Site ◽  
Transcriptional Activation ◽  
Binding Sites ◽  
Binding Protein ◽  
Binding Domain ◽  
Dominant Negative Effect ◽  
Dna Binding Domain ◽  
Wild Type ◽  
Carboxy Terminal

RAP1 is an essential sequence-specific DNA-binding protein in Saccharomyces cerevisiae whose binding sites are found in a large number of promoters, where they function as upstream activation sites, and at the silencer elements of the HMR and HML mating-type loci, where they are important for repression. We have examined the involvement of specific regions of the RAP1 protein in both repression and activation of transcription by studying the properties of a series of hybrid proteins containing RAP1 sequences fused to the DNA-binding domain of the yeast protein GAL4 (amino acids 1 to 147). GAL4 DNA-binding domain/RAP1 hybrids containing only the carboxy-terminal third of the RAP1 protein (which lacks the RAP1 DNA-binding domain) function as transcriptional activators of a reporter gene containing upstream GAL4 binding sites. Expression of some hybrids from the strong ADH1 promoter on multicopy plasmids has a dominant negative effect on silencers, leading to either partial or complete derepression of normally silenced genes. The GAL4/RAP1 hybrids have different effects on wild-type and several mutated but functional silencers. Silencers lacking either an autonomously replicating sequence consensus element or the RAP1 binding site are strongly derepressed, whereas the wild-type silencer or a silencer containing a deletion of the binding site for another silencer-binding protein, ABF1, are only weakly affected by hybrid expression. By examining a series of GAL4 DNA-binding domain/RAP1 hybrids, we have mapped the transcriptional activation and derepression functions to specific parts of the RAP1 carboxy terminus.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Identification of amino acids essential for DNA binding and dimerization in p67SRF: implications for a novel DNA-binding motif

1993 ◽  
Vol 13 (1) ◽  
pp. 123-132
Author(s):  
A D Sharrocks ◽  
H Gille ◽  
P E Shaw
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Dna Binding ◽  
Transcriptional Activation ◽  
Serum Response Factor ◽  
Alpha Helix ◽  
Site Directed Mutagenesis ◽  
Binding Motif ◽  
Amino Acid Peptide ◽  
Serum Response

The serum response factor (p67SRF) binds to a palindromic sequence in the c-fos serum response element (SRE). A second protein, p62TCF binds in conjunction with p67SRF to form a ternary complex, and it is through this complex that growth factor-induced transcriptional activation of c-fos is thought to take place. A 90-amino-acid peptide, coreSRF, is capable for dimerizing, binding DNA, and recruiting p62TCF. By using extensive site-directed mutagenesis we have investigated the role of individual coreSRF amino acids in DNA binding. Mutant phenotypes were defined by gel retardation and cross-linking analyses. Our results have identified residues essential for either DNA binding or dimerization. Three essential basic amino acids whose conservative mutation severely reduced DNA binding were identified. Evidence which is consistent with these residues being on the face of a DNA binding alpha-helix is presented. A phenylalanine residue and a hexameric hydrophobic box are identified as essential for dimerization. The amino acid phasing is consistent with the dimerization interface being presented as a continuous region on a beta-strand. A putative second alpha-helix acts as a linker between these two regions. This study indicates that p67SRF is a member of a protein family which, in common with many DNA binding proteins, utilize an alpha-helix for DNA binding. However, this alpha-helix is contained within a novel domain structure.

A position-dependent transcription-activating domain in TFIIIA

1993 ◽  
Vol 13 (12) ◽  
pp. 7496-7506
Author(s):  
X Mao ◽  
M K Darby
Keyword(s):  
Amino Acids ◽  
Dna Binding ◽  
Rna Polymerase ◽  
Zinc Finger ◽  
Transcriptional Activation ◽  
Transcriptional Activity ◽  
Rna Polymerase Iii ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
5S Rna

Transcription of the Xenopus 5S RNA gene by RNA polymerase III requires the gene-specific factor TFIIIA. To identify domains within TFIIIA that are essential for transcriptional activation, we have expressed C-terminal deletion, substitution, and insertion mutants of TFIIIA in bacteria as fusions with maltose-binding protein (MBP). The MBP-TFIIIA fusion protein specifically binds to the 5S RNA gene internal control region and complements transcription in a TFIIIA-depleted oocyte nuclear extract. Random, cassette-mediated mutagenesis of the carboxyl region of TFIIIA, which is not required for promoter binding, has defined a 14-amino-acid region that is critical for transcriptional activation. In contrast to activators of RNA polymerase II, the activity of the TFIIIA activation domain is strikingly sensitive to its position relative to the DNA-binding domain. When the eight amino acids that separate the transcription-activating domain from the last zinc finger are deleted, transcriptional activity is lost. Surprisingly, diverse amino acids can replace these eight amino acids with restoration of full transcriptional activity, suggesting that the length and not the sequence of this region is important. Insertion of amino acids between the zinc finger region and the transcription-activating domain causes a reduction in transcription proportional to the number of amino acids introduced. We propose that to function, the transcription-activating domain of TFIIIA must be correctly positioned at a minimum distance from the DNA-binding domain.

scholarly journals Evi-1, a murine zinc finger proto-oncogene, encodes a sequence-specific DNA-binding protein.

1991 ◽  
Vol 11 (5) ◽  
pp. 2665-2674 ◽  
Author(s):  
A S Perkins ◽  
R Fishel ◽  
N A Jenkins ◽  
N G Copeland
Keyword(s):  
Dna Binding ◽  
Binding Site ◽  
Zinc Finger ◽  
Sequence Data ◽  
Binding Protein ◽  
Consensus Sequence ◽  
Dna Binding Protein ◽  
Polymerase Chain Reaction Method ◽  
Nucleotide Sequence Data ◽  
Carboxy Terminal

Evi-1 was originally identified as a common site of viral integration in murine myeloid tumors. Evi-1 encodes a 120-kDa polypeptide containing 10 zinc finger motifs located in two domains 380 amino acids apart and an acidic domain located carboxy terminal to the second set of zinc fingers. These features suggest that Evi-1 is a site-specific DNA-binding protein involved in the regulation of RNA transcription. We have purified Evi-1 protein from E. coli and have employed a gel shift-polymerase chain reaction method using random oligonucleotides to identify a high-affinity binding site for Evi-1. The consensus sequence for this binding site is TGACAAGATAA. Evi-1 protein specifically protects this motif from DNase I digestion. By searching the nucleotide sequence data bases, we have found this binding site both in sequences 5' to genes in putative or known regulatory regions and within intron sequences.

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