scholarly journals Two monomers of yeast transcription factor ADR1 bind a palindromic sequence symmetrically to activate ADH2 expression.

1991 ◽  
Vol 11 (3) ◽  
pp. 1566-1577 ◽  
Author(s):  
S K Thukral ◽  
A Eisen ◽  
E T Young
Keyword(s):  
Transcription Factor ◽  
Amino Acid ◽  
Dna Binding ◽  
Dimethyl Sulfate ◽  
Binding Activity ◽  
Single Amino Acid ◽  
Minor Effect ◽  
Palindromic Sequence ◽  
Amino Terminal ◽  
Dna Binding Activity

ADR1 is a transcription factor from Saccharomyces cerevisiae that regulates ADH2 expression through a 22-bp palindromic sequence (UAS1). Size fractionation studies revealed that full-length ADR1 and a truncated ADR1 protein containing the first 229 amino acids, which has the complete DNA-binding domain, ADR1:17-229, exist as monomers in solution. However, two complexes were formed with target DNA-binding sites. UV-cross-linking studies suggested that these two complexes represent one and two molecules of ADR1 bound to DNA. Studies of ADR1 complexes formed with wild-type UAS1, asymmetrically altered UAS1, and one half of UAS1 showed that ADR1 can bind to one half of UAS1 and gives rise to a complex containing one molecule of ADR1. Dimethyl sulfate interference studies were consistent with this interpretation and in addition indicated that purine contact sites in each half of UAS1 were identical. Increasing the distance between the two halves of UAS1 had at most a minor effect of the thermodynamics of formation of the two complexes. These data are more consistent with ADR1 binding as two independent monomers, one to each half of UAS1. However, binding of two ADR1 monomers at UAS1 is apparently essential for transactivation in vivo. Further, we have identified a stretch of 18 amino acid residues amino terminal to the zinc two-finger domains of ADR1 which is essential for DNA-binding activity. Single amino acid substitutions of residues in this region resulted in severely reduced DNA-binding activity.

Two monomers of yeast transcription factor ADR1 bind a palindromic sequence symmetrically to activate ADH2 expression

1991 ◽  
Vol 11 (3) ◽  
pp. 1566-1577
Author(s):  
S K Thukral ◽  
A Eisen ◽  
E T Young
Keyword(s):  
Transcription Factor ◽  
Amino Acid ◽  
Dna Binding ◽  
Dimethyl Sulfate ◽  
Binding Activity ◽  
Single Amino Acid ◽  
Minor Effect ◽  
Palindromic Sequence ◽  
Amino Terminal ◽  
Dna Binding Activity

ADR1 is a transcription factor from Saccharomyces cerevisiae that regulates ADH2 expression through a 22-bp palindromic sequence (UAS1). Size fractionation studies revealed that full-length ADR1 and a truncated ADR1 protein containing the first 229 amino acids, which has the complete DNA-binding domain, ADR1:17-229, exist as monomers in solution. However, two complexes were formed with target DNA-binding sites. UV-cross-linking studies suggested that these two complexes represent one and two molecules of ADR1 bound to DNA. Studies of ADR1 complexes formed with wild-type UAS1, asymmetrically altered UAS1, and one half of UAS1 showed that ADR1 can bind to one half of UAS1 and gives rise to a complex containing one molecule of ADR1. Dimethyl sulfate interference studies were consistent with this interpretation and in addition indicated that purine contact sites in each half of UAS1 were identical. Increasing the distance between the two halves of UAS1 had at most a minor effect of the thermodynamics of formation of the two complexes. These data are more consistent with ADR1 binding as two independent monomers, one to each half of UAS1. However, binding of two ADR1 monomers at UAS1 is apparently essential for transactivation in vivo. Further, we have identified a stretch of 18 amino acid residues amino terminal to the zinc two-finger domains of ADR1 which is essential for DNA-binding activity. Single amino acid substitutions of residues in this region resulted in severely reduced DNA-binding activity.

Synthesis of an enzymatically active FLP recombinase in vitro: search for a DNA-binding domain

1989 ◽  
Vol 9 (5) ◽  
pp. 1987-1995
Author(s):  
A A Amin ◽  
P D Sadowski
Keyword(s):  
Amino Acid ◽  
Dna Binding ◽  
In Vitro Transcription ◽  
Binding Activity ◽  
Binding Domain ◽  
Translation System ◽  
Dna Binding Domain ◽  
Amino Terminal ◽  
Dna Binding Activity

We have used an in vitro transcription and translation system to synthesize an enzymatically active FLP protein. The FLP mRNA synthesized in vitro by SP6 polymerase is translated efficiently in a rabbit reticulocyte lysate to produce enzymatically active FLP. Using this system, we assessed the effect of deletions and tetrapeptide insertions on the ability of the respective variant proteins synthesized in vitro to bind to the FLP recognition target site and to carry out excisive recombination. Deletions of as few as six amino acids from either the carboxy- or amino-terminal region of FLP resulted in loss of binding activity. Likewise, insertions at amino acid positions 79, 203, and 286 abolished DNA-binding activity. On the other hand, a protein with an insertion at amino acid 364 retained significant DNA-binding activity but had no detectable recombination activity. Also, an insertion at amino acid 115 had no measurable effect on DNA binding, but recombination was reduced by 95%. In addition, an insertion at amino acid 411 had no effect on DNA binding and recombination. On the basis of these results, we conclude that this approach fails to define a discrete DNA-binding domain. The possible reasons for this result are discussed.

cDNA cloning of transcription factor E4TF1 subunits with Ets and notch motifs

1993 ◽  
Vol 13 (3) ◽  
pp. 1385-1391
Author(s):  
H Watanabe ◽  
J Sawada ◽  
K Yano ◽  
K Yamaguchi ◽  
M Goto ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Transcription Factors ◽  
Amino Acid ◽  
Dna Binding ◽  
Amino Acid Sequence ◽  
Binding Activity ◽  
Amino Acid Sequences ◽  
Predicted Amino Acid Sequence ◽  
Cdna Clones ◽  
Dna Binding Activity

E4TF1 was originally identified as one of the transcription factors responsible for adenovirus E4 gene transcription. It is composed of two subunits, a DNA binding protein with a molecular mass of 60 kDa and a 53-kDa transcription-activating protein. Heterodimerization of these two subunits is essential for the protein to function as a transcription factor. In this study, we identified a new E4TF1 subunit, designated E4TF1-47, which has no DNA binding activity but can associate with E4TF1-60. We then cloned the cDNAs for each of the E4TF1 subunits. E4TF1 was purified, and the partial amino acid sequence of each subunit was determined. The predicted amino acid sequence of each cDNA clone revealed that E4TF1-60 had an ETS domain, which is a DNA binding domain common to ets-related transcription factors. E4TF1-53 had four tandemly repeated notch-ankyrin motifs. The putative cDNA of E4TF1-47 coded almost the same amino acid sequences as E4TF1-53. Three hundred and thirty-two amino acids of the N termini of E4TF1-47 and -53 were identical except for one amino acid insertion in E4TF1-53, and they differ from each other at the C terminus. These three recombinant cDNA clones were expressed in Escherichia coli, and the proteins behaved in the same manner as purified proteins in a gel retardation assay. Nucleotide and predicted amino acid sequences were highly homologous to GABP-alpha and -beta, which is further supported by the observation that GABP-specific antibody can recognize human E4TF1.

scholarly journals Synthesis of an enzymatically active FLP recombinase in vitro: search for a DNA-binding domain.

1989 ◽  
Vol 9 (5) ◽  
pp. 1987-1995 ◽  
Author(s):  
A A Amin ◽  
P D Sadowski
Keyword(s):  
Amino Acid ◽  
Dna Binding ◽  
In Vitro Transcription ◽  
Binding Activity ◽  
Binding Domain ◽  
Translation System ◽  
Dna Binding Domain ◽  
Amino Terminal ◽  
Dna Binding Activity

We have used an in vitro transcription and translation system to synthesize an enzymatically active FLP protein. The FLP mRNA synthesized in vitro by SP6 polymerase is translated efficiently in a rabbit reticulocyte lysate to produce enzymatically active FLP. Using this system, we assessed the effect of deletions and tetrapeptide insertions on the ability of the respective variant proteins synthesized in vitro to bind to the FLP recognition target site and to carry out excisive recombination. Deletions of as few as six amino acids from either the carboxy- or amino-terminal region of FLP resulted in loss of binding activity. Likewise, insertions at amino acid positions 79, 203, and 286 abolished DNA-binding activity. On the other hand, a protein with an insertion at amino acid 364 retained significant DNA-binding activity but had no detectable recombination activity. Also, an insertion at amino acid 115 had no measurable effect on DNA binding, but recombination was reduced by 95%. In addition, an insertion at amino acid 411 had no effect on DNA binding and recombination. On the basis of these results, we conclude that this approach fails to define a discrete DNA-binding domain. The possible reasons for this result are discussed.

scholarly journals Phenotypic Analysis of Random hnsMutations Differentiate DNA-Binding Activity from Properties offimA Promoter Inversion Modulation and Bacterial Motility

1999 ◽  
Vol 181 (3) ◽  
pp. 941-948 ◽  
Author(s):  
Gina M. Donato ◽  
Thomas H. Kawula
Keyword(s):  
Amino Acid ◽  
Dna Binding ◽  
Constitutive Expression ◽  
Binding Activity ◽  
Single Amino Acid ◽  
Amino Acid Substitutions ◽  
Phenotypic Analysis ◽  
Mutator Strain ◽  
Dna Binding Activity ◽  
And Function

ABSTRACT H-NS is a major Escherichia coli nucleoid-associated protein involved in bacterial DNA condensation and global modulation of gene expression. This protein exists in cells as at least two different isoforms separable by isoelectric focusing. Among other phenotypes, mutations in hns result in constitutive expression of theproU and fimB genes, increased fimApromoter inversion rates, and repression of the flhCDmaster operon required for flagellum biosynthesis. To understand the relationship between H-NS structure and function, we transformed a cloned hns gene into a mutator strain and collected a series of mutant alleles that failed to repress proUexpression. Each of these isolated hns mutant alleles also failed to repress fimB expression, suggesting that H-NS-specific repression of proU and fimBoccurs by similar mechanisms. Conversely, alleles encoding single amino acid substitutions in the C-terminal DNA-binding domain of H-NS resulted in significantly reduced affinity for DNA yet conferred a wild-type fimA promoter inversion frequency, indicating that the mechanism of H-NS activity in modulating promoter inversion is independent of DNA binding. Furthermore, two specific H-NS amino acid substitutions resulted in hypermotile bacteria, while C-terminal H-NS truncations exhibited reduced motility. We also analyzed H-NS isoform composition expressed by various hnsmutations and found that the N-terminal 67 amino acids were sufficient to support posttranslational modification and that substitutions at positions 18 and 26 resulted in the expression of a single H-NS isoform. These results are discussed in terms of H-NS domain organization and implications for biological activity.

scholarly journals cDNA cloning of transcription factor E4TF1 subunits with Ets and notch motifs.

1993 ◽  
Vol 13 (3) ◽  
pp. 1385-1391 ◽  
Author(s):  
H Watanabe ◽  
J Sawada ◽  
K Yano ◽  
K Yamaguchi ◽  
M Goto ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Transcription Factors ◽  
Amino Acid ◽  
Dna Binding ◽  
Amino Acid Sequence ◽  
Binding Activity ◽  
Amino Acid Sequences ◽  
Predicted Amino Acid Sequence ◽  
Cdna Clones ◽  
Dna Binding Activity

E4TF1 was originally identified as one of the transcription factors responsible for adenovirus E4 gene transcription. It is composed of two subunits, a DNA binding protein with a molecular mass of 60 kDa and a 53-kDa transcription-activating protein. Heterodimerization of these two subunits is essential for the protein to function as a transcription factor. In this study, we identified a new E4TF1 subunit, designated E4TF1-47, which has no DNA binding activity but can associate with E4TF1-60. We then cloned the cDNAs for each of the E4TF1 subunits. E4TF1 was purified, and the partial amino acid sequence of each subunit was determined. The predicted amino acid sequence of each cDNA clone revealed that E4TF1-60 had an ETS domain, which is a DNA binding domain common to ets-related transcription factors. E4TF1-53 had four tandemly repeated notch-ankyrin motifs. The putative cDNA of E4TF1-47 coded almost the same amino acid sequences as E4TF1-53. Three hundred and thirty-two amino acids of the N termini of E4TF1-47 and -53 were identical except for one amino acid insertion in E4TF1-53, and they differ from each other at the C terminus. These three recombinant cDNA clones were expressed in Escherichia coli, and the proteins behaved in the same manner as purified proteins in a gel retardation assay. Nucleotide and predicted amino acid sequences were highly homologous to GABP-alpha and -beta, which is further supported by the observation that GABP-specific antibody can recognize human E4TF1.

scholarly journals DNA-Binding Activity of Amino-Terminal Domains of the Bacillus subtilis AbrB Protein

2001 ◽  
Vol 183 (13) ◽  
pp. 4094-4098 ◽  
Author(s):  
Ke Xu ◽  
Mark A. Strauch
Keyword(s):  
Amino Acid ◽  
Dna Binding ◽  
Binding Activity ◽  
Amino Acid Residues ◽  
Disulfide Bonding ◽  
Amino Terminal ◽  
Dna Binding Activity ◽  
Dna Binding Specificity ◽  
Dna Binding Affinity ◽  
Truncated Variants

ABSTRACT Two truncated variants of AbrB, comprising either its first 53 (AbrBN53) or first 55 (AbrBN55) amino acid residues, were constructed and purified. Noncovalently linked homodimers of the truncated variants exhibited very weak DNA-binding activity. Cross-linking AbrBN55 dimers into tetramers and higher-order multimers (via disulfide bonding between penultimate cysteine residues) resulted in proteins having DNA-binding affinity comparable to and DNA-binding specificity identical to those of intact, wild-type AbrB. These results indicate that the DNA recognition and specificity determinants of AbrB binding lie solely within its N-terminal amino acid sequence.

A single polypeptide possesses the binding and transcription activities of the adenovirus major late transcription factor

1986 ◽  
Vol 6 (12) ◽  
pp. 4723-4733
Author(s):  
L A Chodosh ◽  
R W Carthew ◽  
P A Sharp
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
Binding Site ◽  
Rna Polymerase Ii ◽  
Sodium Dodecyl ◽  
Binding Activity ◽  
Affinity Column ◽  
Dna Binding Activity ◽  
Dna Fragment

A simple approach has been developed for the unambiguous identification and purification of sequence-specific DNA-binding proteins solely on the basis of their ability to bind selectively to their target sequences. Four independent methods were used to identify the promoter-specific RNA polymerase II transcription factor MLTF as a 46-kilodalton (kDa) polypeptide. First, a 46-kDa protein was specifically cross-linked by UV irradiation to a body-labeled DNA fragment containing the MLTF binding site. Second, MLTF sedimented through glycerol gradients at a rate corresponding to a protein of native molecular weight 45,000 to 50,000. Third, a 46-kDa protein was specifically retained on a biotin-streptavidin matrix only when the DNA fragment coupled to the matrix contained the MLTF binding site. Finally, proteins from the most highly purified fraction which were eluted and renatured from the 44- to 48-kDa region of a sodium dodecyl sulfate-polyacrylamide gel exhibited both binding and transcription-stimulatory activities. The DNA-binding activity was purified 100,000-fold by chromatography through three conventional columns plus a DNA affinity column. Purified MLTF was characterized with respect to the kinetic and thermodynamic properties of DNA binding. These parameters indicate a high degree of occupancy of MLTF binding sites in vivo.

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