scholarly journals Analysis of DNA binding by the adenovirus type 5 E1A oncoprotein

2002 ◽  
Vol 83 (3) ◽  
pp. 517-524 ◽  
Author(s):  
Nikita Avvakumov ◽  
Majdina Sahbegovic ◽  
Zhiying Zhang ◽  
Michael Shuen ◽  
Joe S. Mymryk
Keyword(s):  
Amino Acids ◽  
Dna Binding ◽  
Transcriptional Activation ◽  
Adenovirus Type ◽  
Dna Binding Domain ◽  
Activation Domain ◽  
Transcriptional Activation Domain ◽  
Adenovirus Type 5

Adenovirus type 5 E1A proteins interact with cellular regulators of transcription to reprogram gene expression in the infected or transformed cell. Although E1A also interacts with DNA directly in vitro, it is not clear how this relates to its function in vivo. The N-terminal conserved regions 1, 2 and 3 and the C-terminal portions of E1A were prepared as purified recombinant proteins and analyses showed that only the C-terminal region bound DNA in vitro. Deletion of E1A amino acids 201–220 inhibited binding and a minimal fragment encompassing amino acids 201–218 of E1A was sufficient for binding single- and double-stranded DNA. This portion of E1A also bound the cation-exchange resins cellulose phosphate and carboxymethyl Sepharose. As this region contains six basic amino acids, in vitro binding of E1A to DNA probably results from an ionic interaction with the phosphodiester backbone of DNA. Studies in Saccharomyces cerevisiae have shown that expression of a strong transcriptional activation domain fused to a DNA-binding domain can inhibit growth. Although fusion of the C-terminal region of E1A to a strong transcriptional activation domain inhibited growth when expressed in yeast, this was not mediated by the DNA-binding domain identified in vitro. These data suggest that E1A does not bind DNA in vivo.

Reconstitution of transcriptional activation domains by reiteration of short peptide segments reveals the modular organization of a glutamine-rich activation domain

1994 ◽  
Vol 14 (9) ◽  
pp. 6056-6067
Author(s):  
M Tanaka ◽  
W Herr
Keyword(s):  
Dna Binding ◽  
Transcriptional Activation ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Activation Domain ◽  
Activation Domains ◽  
Transcriptional Activation Domain ◽  
Pou Domain

The POU domain activator Oct-2 contains an N-terminal glutamine-rich transcriptional activation domain. An 18-amino-acid segment (Q18III) from this region reconstituted a fully functional activation domain when tandemly reiterated and fused to either the Oct-2 or GAL4 DNA-binding domain. A minimal transcriptional activation domain likely requires three tandem Q18III segments, because one or two tandem Q18III segments displayed little activity, whereas three to five tandem segments were active and displayed increasing activity with increasing copy number. As with natural Oct-2 activation domains, in our assay a reiterated activation domain required a second homologous or heterologous activation domain to stimulate transcription effectively when fused to the Oct-2 POU domain. These results suggest that there are different levels of synergy within and among activation domains. Analysis of reiterated activation domains containing mutated Q18III segments revealed that leucines and glutamines, but not serines or threonines, are critical for activity in vivo. Curiously, several reiterated activation domains that were inactive in vivo were active in vitro, suggesting that there are significant functional differences in our in vivo and in vitro assays. Reiteration of a second 18-amino-acid segment from the Oct-2 glutamine-rich activation domain (Q18II) was also active, but its activity was DNA-binding domain specific, because it was active when fused to the GAL4 than to the Oct-2 DNA-binding domain. The ability of separate short peptide segments derived from a single transcriptional activation domain to activate transcription after tandem reiteration emphasizes the flexible and modular nature of a transcriptional activation domain.

scholarly journals Reconstitution of transcriptional activation domains by reiteration of short peptide segments reveals the modular organization of a glutamine-rich activation domain.

1994 ◽  
Vol 14 (9) ◽  
pp. 6056-6067 ◽  
Author(s):  
M Tanaka ◽  
W Herr
Keyword(s):  
Dna Binding ◽  
Transcriptional Activation ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Activation Domain ◽  
Activation Domains ◽  
Transcriptional Activation Domain ◽  
Pou Domain

The POU domain activator Oct-2 contains an N-terminal glutamine-rich transcriptional activation domain. An 18-amino-acid segment (Q18III) from this region reconstituted a fully functional activation domain when tandemly reiterated and fused to either the Oct-2 or GAL4 DNA-binding domain. A minimal transcriptional activation domain likely requires three tandem Q18III segments, because one or two tandem Q18III segments displayed little activity, whereas three to five tandem segments were active and displayed increasing activity with increasing copy number. As with natural Oct-2 activation domains, in our assay a reiterated activation domain required a second homologous or heterologous activation domain to stimulate transcription effectively when fused to the Oct-2 POU domain. These results suggest that there are different levels of synergy within and among activation domains. Analysis of reiterated activation domains containing mutated Q18III segments revealed that leucines and glutamines, but not serines or threonines, are critical for activity in vivo. Curiously, several reiterated activation domains that were inactive in vivo were active in vitro, suggesting that there are significant functional differences in our in vivo and in vitro assays. Reiteration of a second 18-amino-acid segment from the Oct-2 glutamine-rich activation domain (Q18II) was also active, but its activity was DNA-binding domain specific, because it was active when fused to the GAL4 than to the Oct-2 DNA-binding domain. The ability of separate short peptide segments derived from a single transcriptional activation domain to activate transcription after tandem reiteration emphasizes the flexible and modular nature of a transcriptional activation domain.

scholarly journals Identification of transcriptional activation and inhibitory domains in serum response factor (SRF) by using GAL4-SRF constructs

1993 ◽  
Vol 13 (8) ◽  
pp. 4640-4647
Author(s):  
F E Johansen ◽  
R Prywes
Keyword(s):  
Amino Acids ◽  
Dna Binding ◽  
Transcriptional Activation ◽  
Serum Response Factor ◽  
Binding Domain ◽  
Response Factor ◽  
Dna Binding Domain ◽  
Activation Domain ◽  
Transcriptional Activation Domain ◽  
Serum Response

The binding of serum response factor (SRF) to the c-fos serum response element has been shown to be essential for serum and growth factor activation of c-Fos. Since SRF is ubiquitously expressed, it has been difficult to measure the activity of SRF introduced into cells. To assay for functions of SRF in cells, we have changed its DNA binding specificity by fusing it to the DNA binding domain of GAL4. Transfection of GAL4-SRF constructs into cells has allowed us to identify SRF's transcriptional activation domain as well as domains which inhibit this activity. First, we found that the transcriptional activation domain maps to between amino acids 339 and 508 in HeLa cells and to between amino acids 414 and 508 in NIH 3T3 cells. Second, we show that in the context of GAL4-SRF constructs, there are two separate domains of SRF that can inhibit its activation domain. Although these domains overlap the DNA binding and dimerization domains of SRF, these functions were not required for inhibition. Finally, we show that one of the inhibitory domains is modular in that it can also inhibit activation when it is moved amino terminal to GAL4's DNA binding domain in an SRF-GAL4-SRF construct. The implications of these inhibitory domains for SRF regulation are discussed.

scholarly journals Identification of transcriptional activation and inhibitory domains in serum response factor (SRF) by using GAL4-SRF constructs.

1993 ◽  
Vol 13 (8) ◽  
pp. 4640-4647 ◽  
Author(s):  
F E Johansen ◽  
R Prywes
Keyword(s):  
Amino Acids ◽  
Dna Binding ◽  
Transcriptional Activation ◽  
Serum Response Factor ◽  
Binding Domain ◽  
Response Factor ◽  
Dna Binding Domain ◽  
Activation Domain ◽  
Transcriptional Activation Domain ◽  
Serum Response

The binding of serum response factor (SRF) to the c-fos serum response element has been shown to be essential for serum and growth factor activation of c-Fos. Since SRF is ubiquitously expressed, it has been difficult to measure the activity of SRF introduced into cells. To assay for functions of SRF in cells, we have changed its DNA binding specificity by fusing it to the DNA binding domain of GAL4. Transfection of GAL4-SRF constructs into cells has allowed us to identify SRF's transcriptional activation domain as well as domains which inhibit this activity. First, we found that the transcriptional activation domain maps to between amino acids 339 and 508 in HeLa cells and to between amino acids 414 and 508 in NIH 3T3 cells. Second, we show that in the context of GAL4-SRF constructs, there are two separate domains of SRF that can inhibit its activation domain. Although these domains overlap the DNA binding and dimerization domains of SRF, these functions were not required for inhibition. Finally, we show that one of the inhibitory domains is modular in that it can also inhibit activation when it is moved amino terminal to GAL4's DNA binding domain in an SRF-GAL4-SRF construct. The implications of these inhibitory domains for SRF regulation are discussed.

scholarly journals Extensive mutagenesis of a transcriptional activation domain identifies single hydrophobic and acidic amino acids important for activation in vivo.

1997 ◽  
Vol 17 (1) ◽  
pp. 115-122 ◽  
Author(s):  
M B Sainz ◽  
S A Goff ◽  
V L Chandler
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Transcriptional Activation ◽  
Carboxyl Terminus ◽  
Wild Type ◽  
Activation Domain ◽  
Hydrophobic Residues ◽  
Transcriptional Activation Domain ◽  
Acidic Amino Acids

C1 is a transcriptional activator of genes encoding biosynthetic enzymes of the maize anthocyanin pigment pathway. C1 has an amino terminus homologous to Myb DNA-binding domains and an acidic carboxyl terminus that is a transcriptional activation domain in maize and yeast cells. To identify amino acids critical for transcriptional activation, an extensive random mutagenesis of the C1 carboxyl terminus was done. The C1 activation domain is remarkably tolerant of amino acid substitutions, as changes at 34 residues had little or no effect on transcriptional activity. These changes include introduction of helix-incompatible amino acids throughout the C1 activation domain and alteration of most single acidic amino acids, suggesting that a previously postulated amphipathic alpha-helix is not required for activation. Substitutions at two positions revealed amino acids important for transcriptional activation. Replacement of leucine 253 with a proline or glutamine resulted in approximately 10% of wild-type transcriptional activation. Leucine 253 is in a region of C1 in which several hydrophobic residues align with residues important for transcriptional activation by the herpes simplex virus VP16 protein. However, changes at all other hydrophobic residues in C1 indicate that none are critical for C1 transcriptional activation. The other important amino acid in C1 is aspartate 262, as a change to valine resulted in only 24% of wild-type transcriptional activation. Comparison of our C1 results with those from VP16 reveal substantial differences in which amino acids are required for transcriptional activation in vivo by these two acidic activation domains.

scholarly journals Synergistic transcriptional activation by CTF/NF-I and the estrogen receptor involves stabilized interactions with a limiting target factor.

1991 ◽  
Vol 11 (6) ◽  
pp. 2937-2945 ◽  
Author(s):  
E Martinez ◽  
Y Dusserre ◽  
W Wahli ◽  
N Mermod
Keyword(s):  
Estrogen Receptor ◽  
Dna Binding ◽  
Transcriptional Activation ◽  
Transcription Initiation ◽  
Limiting Factor ◽  
Gene Promoters ◽  
Protein Coding ◽  
Transcriptional Activation Domain

Transcription initiation at eukaryotic protein-coding gene promoters is regulated by a complex interplay of site-specific DNA-binding proteins acting synergistically or antagonistically. Here, we have analyzed the mechanisms of synergistic transcriptional activation between members of the CCAAT-binding transcription factor/nuclear factor I (CTF/NF-I) family and the estrogen receptor. By using cotransfection experiments with HeLa cells, we show that the proline-rich transcriptional activation domain of CTF-1, when fused to the GAL4 DNA-binding domain, synergizes with each of the two estrogen receptor-activating regions. Cooperative DNA binding between the GAL4-CTF-1 fusion and the estrogen receptor does not occur in vitro, and in vivo competition experiments demonstrate that both activators can be specifically inhibited by the overexpression of a proline-rich competitor, indicating that a common limiting factor is mediating their transcriptional activation functions. Furthermore, the two activators functioning synergistically are much more resistant to competition than either factor alone, suggesting that synergism between CTF-1 and the estrogen receptor is the result of a stronger tethering of the limiting target factor(s) to the two promoter-bound activators.

scholarly journals Characterization of Neurospora CPC1, a bZIP DNA-binding protein that does not require aligned heptad leucines for dimerization.

1991 ◽  
Vol 11 (2) ◽  
pp. 935-944 ◽  
Author(s):  
J L Paluh ◽  
C Yanofsky
Keyword(s):  
Amino Acid ◽  
Dna Binding ◽  
Transcriptional Activation ◽  
Transcription Activator ◽  
Dimerization Domain ◽  
Activation Domain ◽  
Band Shift ◽  
Transcriptional Activation Domain ◽  
Chimeric Polypeptide

CPC1 is the transcriptional activator of amino acid biosynthetic genes of Neurospora crassa. CPC1 function in vivo was abolished upon deletion of segments of cpc-1 corresponding to the presumed transcription activation domain, the DNA-binding and dimerization domains, or a 52-residue connector segment of CPC1. A truncated CPC1 polypeptide containing only the carboxy-terminal 57-residue segment of CPC1 was sufficient to form homodimers that bound DNA. However, deletion of the segment of cpc-1 corresponding to the connector segment in the full-length CPC1 polypeptide abolished DNA binding. Removal of a segment of cpc-1 corresponding to the GIn-rich region of CPC1 reduced in vivo function only slightly. The homologous transcription activator of Saccharomyces cerevisiae, GCN4, did not substitute for CPC1 in N. crassa. Chimeric CPC1-GCN4 polypeptides that contained the GCN4 transcriptional activation domain or the domain of GCN4 that corresponds to the essential 52-residue connector segment of CPC1, functioned with reduced efficiency. However, a chimeric polypeptide containing the GCN4 DNA-binding and dimerization domains in place of those of CPC1 functioned essentially as well as wild-type CPC1. The basic and dimerization domains of CPC1 were characterized by introducing deletions or site-directed amino acid replacements. The basic region was required for DNA binding but not for dimerization. CPC1 has a short dimerization domain containing heptad residues Leu-1, Leu-2, Trp-3, and His-4. When Val was substituted for Leu-1 or Leu-2, CPC1 was fully active, but when Val replaced Trp-3, dimerization and DNA binding were prevented. DNA band shift analyses with CPC1 heterodimers demonstrated that CPC1 does not require aligned heptad leucine residues for dimerization. Replacement of two charged residues located between Leu-1 and Leu-2 of CPC1 abolished dimerization and DNA binding.

scholarly journals Sex-specific and non-sex-specific oligomerization domains in both of the doublesex transcription factors from Drosophila melanogaster.

1996 ◽  
Vol 16 (6) ◽  
pp. 3106-3111 ◽  
Author(s):  
W An ◽  
S Cho ◽  
H Ishii ◽  
P C Wensink
Keyword(s):  
Amino Acids ◽  
Drosophila Melanogaster ◽  
Transcription Factors ◽  
Dna Binding ◽  
Transcriptional Activation ◽  
Yolk Protein ◽  
Female Specific ◽  
Oligomerization Domain

The doublesex gene of Drosophila melanogaster encodes the alternatively spliced, sex-specific transcription factors DSXM and DSXF. These factors regulate male- and female-specific transcription of many genes. For example, female-specific transcription of the yolk protein 1 gene is regulated by DSXM repression in males and DSXF activation in females. In this study we used in vitro interaction assays and the in vivo yeast two-hybrid method to identify and examine oligomerization domains of the DSX proteins. A 66-amino-acid segment common to both proteins (amino acids 39 to 104) contains a sequence-specific DNA binding domain and an oligomerization domain (OD1). The OD1 domain oligomerizes up to at least a pentamer, but only dimers bound to a palindromic regulatory site in the yolk protein 1 gene are detected. Both subunits of the OD1 dimer are in contact with DNA. Another segment of each protein (amino acids 350 to 412 for DSXF and 350 to 427 for DSXM) contains a second oligomerization domain (OD2F and OD2M, respectively). The OD2 domains have both sex-specific and non-sex-specific sequences which are necessary for oligomerization. On the basis of sequence analysis, we predict that OD2 oligomerizes through coiled-coil interactions. We speculate that the common function of OD1 and OD2 is to oligomerize the full-length proteins, whereas their specialized functions are to form a dimeric DNA binding unit and a sex-specific transcriptional activation or repression unit.

Synergistic transcriptional activation by CTF/NF-I and the estrogen receptor involves stabilized interactions with a limiting target factor

1991 ◽  
Vol 11 (6) ◽  
pp. 2937-2945
Author(s):  
E Martinez ◽  
Y Dusserre ◽  
W Wahli ◽  
N Mermod
Keyword(s):  
Estrogen Receptor ◽  
Dna Binding ◽  
Transcriptional Activation ◽  
Transcription Initiation ◽  
Limiting Factor ◽  
Gene Promoters ◽  
Protein Coding ◽  
Transcriptional Activation Domain

Transcription initiation at eukaryotic protein-coding gene promoters is regulated by a complex interplay of site-specific DNA-binding proteins acting synergistically or antagonistically. Here, we have analyzed the mechanisms of synergistic transcriptional activation between members of the CCAAT-binding transcription factor/nuclear factor I (CTF/NF-I) family and the estrogen receptor. By using cotransfection experiments with HeLa cells, we show that the proline-rich transcriptional activation domain of CTF-1, when fused to the GAL4 DNA-binding domain, synergizes with each of the two estrogen receptor-activating regions. Cooperative DNA binding between the GAL4-CTF-1 fusion and the estrogen receptor does not occur in vitro, and in vivo competition experiments demonstrate that both activators can be specifically inhibited by the overexpression of a proline-rich competitor, indicating that a common limiting factor is mediating their transcriptional activation functions. Furthermore, the two activators functioning synergistically are much more resistant to competition than either factor alone, suggesting that synergism between CTF-1 and the estrogen receptor is the result of a stronger tethering of the limiting target factor(s) to the two promoter-bound activators.

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