scholarly journals Molecular and genetic analysis of the toxic effect of RAP1 overexpression in yeast.

Genetics ◽  
1995 ◽  
Vol 141 (4) ◽  
pp. 1253-1262 ◽  
Author(s):  
K Freeman ◽  
M Gwadz ◽  
D Shore
Keyword(s):  
Dna Binding ◽  
Genetic Analysis ◽  
Toxic Effect ◽  
Transcriptional Activation ◽  
Ribosomal Proteins ◽  
Regulatory Protein ◽  
Point Mutations ◽  
Binding Domain ◽  
Dna Binding Domain

Abstract Rap1p is a context-dependent regulatory protein in yeast that functions as a transcriptional activator of many essential genes, including those encoding ribosomal proteins and glycolytic enzymes. Rap1p also participates in transcriptional silencing at HM mating-type loci and telomeres. Overexpression of RAP1 strongly inhibits cell growth, perhaps by interfering with essential transcriptional activation functions within the cell. Here we report a molecular and genetic analysis of the toxic effect of RAP1 overexpression. We show that toxicity does not require the previously defined Rap1p activation and silencing domains, but instead is dependent upon the DNA-binding domain and an adjacent region of unknown function. Point mutations were identified in the DNA-binding domain that relieve the toxic effect of overexpression. Two of these mutations can complement a RAP1 deletion yet cause growth defects and altered DNA-binding properties in vitro. However, a small deletion of the adjacent (downstream) region that abolishes overexpression toxicity has, by itself, no apparent effect on growth or DNA binding. SKO1/ACR1, which encodes a CREB-like repressor protein in yeast, was isolated as a high copy suppressor of the toxicity caused by RAP1 overexpression. Models related to the regulation of Rap1p activity are discussed.

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 Involvement of the Multidomain Regulatory Protein XynR in Positive Control of Xylanase Gene Expression in the Ruminal Anaerobe Prevotella bryantii B14

2003 ◽  
Vol 185 (7) ◽  
pp. 2219-2226 ◽  
Author(s):  
Kohji Miyazaki ◽  
Hiroyuki Miyamoto ◽  
Derry K. Mercer ◽  
Tatsuaki Hirase ◽  
Jennifer C. Martin ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Binding ◽  
Transmembrane Domain ◽  
Regulatory Protein ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Xylanase Gene ◽  
Prevotella Bryantii ◽  
Genes Encoding

ABSTRACT The xylanase gene cluster from the rumen anaerobe Prevotella bryantii B14 was found to include a gene (xynR) that encodes a multidomain regulatory protein and is downstream from the xylanase and β-xylosidase genes xynA and xynB. Additional genes identified upstream of xynA and xynB include xynD, which encodes an integral membrane protein that has homology with Na:solute symporters; xynE, which is related to the genes encoding acylhydrolases and arylesterases; and xynF, which has homology with the genes encoding α-glucuronidases. XynR includes, in a single 833-amino-acid polypeptide, a putative input domain unrelated to other database sequences, a likely transmembrane domain, histidine kinase motifs, response regulator sequences, and a C-terminal AraC-type helix-turn-helix DNA binding domain. Two transcripts (3.7 and 5.8 kb) were detected with a xynA probe, and the start site of the 3.7-kb transcript encoding xynABD was mapped to a position upstream of xynD. The DNA binding domain of XynR was purified after amplification and overexpression in Escherichia coli and was found to bind to a 141-bp DNA fragment from the region immediately upstream of xynD. In vitro transcription assays demonstrated that XynR stimulates transcription of the 3.7-kb transcript. We concluded that XynR acts as a positive regulator that activates expression of xynABD in P. bryantii B14. This is the first regulatory protein that demonstrates significant homology with the two-component regulatory protein superfamily and has been shown to be involved in the regulation of polysaccharidase gene expression.

scholarly journals Role of cysteine residues in regulation of p53 function.

1995 ◽  
Vol 15 (7) ◽  
pp. 3892-3903 ◽  
Author(s):  
R Rainwater ◽  
D Parks ◽  
M E Anderson ◽  
P Tegtmeyer ◽  
K Mann
Keyword(s):  
Dna Binding ◽  
Transcriptional Activation ◽  
Redox Regulation ◽  
Binding Domain ◽  
Zinc Binding ◽  
Cysteine Residues ◽  
Dna Binding Domain ◽  
Site Specific ◽  
Functional Consequences

Previous studies of p53 have implicated cysteine residues in site-specific DNA binding via zinc coordination and redox regulation (P. Hainaut and J. Milner, Cancer Res. 53:4469-4473, 1993; T. R. Hupp, D. W. Meek, C. A. Midgley, and D. P. Lane, Nucleic Acids Res. 21:3167-3174, 1993). We show here that zinc binding and redox regulation are, at least in part, distinct determinants of the binding of p53 to DNA. Moreover, by substituting serine for each cysteine in murine p53, we have investigated the roles of individual cysteines in the regulation of p53 function. Substitution of serine for cysteine at position 40, 179, 274, 293, or 308 had little or no effect on p53 function. In contrast, replacement of cysteine at position 173, 235, or 239 markedly reduced in vitro DNA binding, completely blocked transcriptional activation, and led to a striking enhancement rather than a suppression of transformation by p53. These three cysteines have been implicated in zinc binding by X-ray diffraction studies (Y. Cho, S. Gorina, P.D. Jeffrey, and N.P. Pavletich, Science 265:346-355, 1994); our studies demonstrate the functional consequences of the inability of the central DNA-binding domain of p53 to studies demonstrate the functional consequences of the inability of the central DNA-binding domain of p53 to bind zinc. Lastly, substitutions for cysteines at position 121, 132, 138, or 272 partially blocked both transactivation and the suppression of transformation by p53. These four cysteines are located in the loop-sheet-helix region of the site-specific DNA-binding domain of p53. Like the cysteines in the zinc-binding region, therefore, these cysteines may cooperate to modulate the structure of the DNA-binding domain. Our findings argue that p53 is subject to more than one level of conformational modulation through oxidation-reduction of cysteines at or near the p53-DNA interface.

scholarly journals Overproduction of v-Myc in the nucleus and its excess over Max are not required for avian fibroblast transformation

1993 ◽  
Vol 13 (6) ◽  
pp. 3623-3631
Author(s):  
A T Tikhonenko ◽  
A R Hartman ◽  
M L Linial
Keyword(s):  
Dna Binding ◽  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Point Mutations ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Localization Signal ◽  
Infected Cells ◽  
Fibroblast Transformation

The cellular proto-oncogene c-myc can acquire transforming potential by a number of different means, including retroviral transduction. The transduced allele generally contains point mutations relative to c-myc and is overexpressed in infected cells, usually as a v-Gag-Myc fusion protein. Upon synthesis, v-Gag-Myc enters the nucleus, forms complexes with its heterodimeric partner Max, and in this complex binds to DNA in a sequence-specific manner. To delineate the role for each of these events in fibroblast transformation, we introduced several mutations into the myc gene of the avian retrovirus MC29. We observed that Gag-Myc with a mutated nuclear localization signal is confined predominantly in the cytoplasm and only about 5% of the protein could be detected in the nucleus (less than the amount of endogenous c-Myc). Consequently, only a small fraction of Max is associated with Myc. However, cells infected with this mutant exhibit a completely transformed phenotype in vitro, suggesting that production of enough v-Gag-Myc to tie up all cellular Max is not needed for transformation. While the nuclear localization signal is dispensable for transformation, minimal changes in the v-Gag-Myc DNA-binding domain completely abolish its transforming potential, consistent with a role of Myc as a transcriptional regulator. One of its potential targets might be the endogenous c-myc, which is repressed in wild-type MC29-infected cells. Our experiments with MC29 mutants demonstrate that c-myc down-regulation depends on the integrity of the v-Myc DNA-binding domain and occurs at the RNA level. Hence, it is conceivable that v-Gag-Myc, either directly or circuitously, regulates c-myc transcription.

scholarly journals Overproduction of v-Myc in the nucleus and its excess over Max are not required for avian fibroblast transformation.

1993 ◽  
Vol 13 (6) ◽  
pp. 3623-3631 ◽  
Author(s):  
A T Tikhonenko ◽  
A R Hartman ◽  
M L Linial
Keyword(s):  
Dna Binding ◽  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Point Mutations ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Localization Signal ◽  
Infected Cells ◽  
Fibroblast Transformation

The cellular proto-oncogene c-myc can acquire transforming potential by a number of different means, including retroviral transduction. The transduced allele generally contains point mutations relative to c-myc and is overexpressed in infected cells, usually as a v-Gag-Myc fusion protein. Upon synthesis, v-Gag-Myc enters the nucleus, forms complexes with its heterodimeric partner Max, and in this complex binds to DNA in a sequence-specific manner. To delineate the role for each of these events in fibroblast transformation, we introduced several mutations into the myc gene of the avian retrovirus MC29. We observed that Gag-Myc with a mutated nuclear localization signal is confined predominantly in the cytoplasm and only about 5% of the protein could be detected in the nucleus (less than the amount of endogenous c-Myc). Consequently, only a small fraction of Max is associated with Myc. However, cells infected with this mutant exhibit a completely transformed phenotype in vitro, suggesting that production of enough v-Gag-Myc to tie up all cellular Max is not needed for transformation. While the nuclear localization signal is dispensable for transformation, minimal changes in the v-Gag-Myc DNA-binding domain completely abolish its transforming potential, consistent with a role of Myc as a transcriptional regulator. One of its potential targets might be the endogenous c-myc, which is repressed in wild-type MC29-infected cells. Our experiments with MC29 mutants demonstrate that c-myc down-regulation depends on the integrity of the v-Myc DNA-binding domain and occurs at the RNA level. Hence, it is conceivable that v-Gag-Myc, either directly or circuitously, regulates c-myc transcription.

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 Cell Cycle Localization, Dimerization, and Binding Domain Architecture of the Telomere Protein cPot1

2004 ◽  
Vol 24 (5) ◽  
pp. 2091-2102 ◽  
Author(s):  
Chao Wei ◽  
Carolyn M. Price
Keyword(s):  
Cell Cycle ◽  
Dna Binding ◽  
Binding Site ◽  
Domain Architecture ◽  
Binding Domain ◽  
Binding Motif ◽  
Dna Binding Domain ◽  
Telomere Protein ◽  
Ob Fold

ABSTRACT Pot1 is a single-stranded-DNA-binding protein that recognizes telomeric G-strand DNA. It is essential for telomere capping in Saccharomyces pombe and regulates telomere length in humans. Human Pot1 also interacts with proteins that bind the duplex region of the telomeric tract. Thus, like Cdc13 from S. cerevisiae, Pot 1 may have multiple roles at the telomere. We show here that endogenous chicken Pot1 (cPot1) is present at telomeres during periods of the cell cycle when t loops are thought to be present. Since cPot1 can bind internal loops and directly adjacent DNA-binding sites, it is likely to fully coat and protect both G-strand overhangs and the displaced G strand of a t loop. The minimum binding site of cPot1 is double that of the S. pombe DNA-binding domain. Although cPot can self associate, dimerization is not required for DNA binding and hence does not explain the binding-site duplication. Instead, the DNA-binding domain appears to be extended to contain a second binding motif in addition to the conserved oligonucleotide-oligosaccharide (OB) fold present in other G-strand-binding proteins. This second motif could be another OB fold. Although dimerization is inefficient in vitro, it may be regulated in vivo and could promote association with other telomere proteins and/or telomere compaction.

scholarly journals Role of GCR2 in transcriptional activation of yeast glycolytic genes.

1992 ◽  
Vol 12 (9) ◽  
pp. 3834-3842 ◽  
Author(s):  
H Uemura ◽  
Y Jigami
Keyword(s):  
Dna Binding ◽  
Fusion Protein ◽  
Transcriptional Activation ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Gene Products ◽  
Lacz Expression ◽  
Genetic Method ◽  
Potential Interactions

The Saccharomyces cerevisiae GCR2 gene affects expression of most of the glycolytic genes. We report the nucleotide sequence of GCR2, which can potentially encode a 58,061-Da protein. There is a small cluster of asparagines near the center and a C-terminal region that would be highly charged but overall neutral. Fairly homologous regions were found between Gcr2 and Gcr1 proteins. To test potential interactions, the genetic method of S. Fields and O. Song (Nature [London] 340:245-246, 1989), which uses protein fusions of candidate gene products with, respectively, the N-terminal DNA-binding domain of Gal4 and the C-terminal activation domain II, assessing restoration of Gal4 function, was used. In a delta gal4 delta gal80 strain, double transformation by plasmids containing, respectively, a Gal4 (transcription-activating region)/Gcr1 fusion and a Gal4 (DNA-binding domain)/Gcr2 fusion activated lacZ expression from an integrated GAL1/lacZ fusion, indicating reconstitution of functional Gal4 through the interaction of Gcr1 and Gcr2 proteins. The Gal4 (transcription-activating region)/Gcr1 fusion protein alone complemented the defects of both gcr1 and gcr2 strains. Furthermore, a Rap1/Gcr2 fusion protein partially complemented the defects of gcr1 strains. These results suggest that Gcr2 has transcriptional activation activity and that the GCR1 and GCR2 gene products function together.

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 An Inhibitory Region of the DNA-Binding Domain of Thyroid Hormone Receptor Blocks Hormone-Dependent Transactivation

1998 ◽  
Vol 12 (1) ◽  
pp. 34-44 ◽  
Author(s):  
Ying Liu ◽  
Akira Takeshita ◽  
Takashi Nagaya ◽  
Aria Baniahmad ◽  
William W. Chin ◽  
...  
Keyword(s):  
Thyroid Hormone ◽  
Dna Binding ◽  
Ligand Binding ◽  
Transcriptional Activation ◽  
Thyroid Hormone Receptor ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Chimeric Receptor ◽  
Receptor System ◽  
Inhibitory Region

Abstract We have employed a chimeric receptor system in which we cotransfected yeast GAL4 DNA-binding domain/retinoid X receptor β ligand-binding domain chimeric receptor (GAL4RXR), thyroid hormone receptor-β (TRβ), and upstream activating sequence-reporter plasmids into CV-1 cells to study repression, derepression, and transcriptional activation. In the absence of T3, unliganded TR repressed transcription to 20% of basal level, and in the presence of T3, liganded TRβ derepressed transcription to basal level. Using this system and a battery of TRβ mutants, we found that TRβ/RXR heterodimer formation is necessary and sufficient for basal repression and derepression in this system. Additionally, an AF-2 domain mutant (E457A) mediated basal repression but not derepression, suggesting that interaction with a putative coactivator at this site may be critical for derepression. Interestingly, a mutant containing only the TRβ ligand binding domain (LBD) not only mediated derepression, but also stimulated transcriptional activation 10-fold higher than basal level. Studies using deletion and domain swap mutants localized an inhibitory region to the TRβ DNA-binding domain. Titration studies further suggested that allosteric changes promoting interaction with coactivators may account for enhanced transcriptional activity by LBD. In summary, our findings suggest that TR heterodimer formation with RXR is important for repression and derepression, and coactivator interaction with the AF-2 domain may be needed for derepression in this chimeric system. Additionally, there may be an inhibitory region in the DNA-binding domain, which reduces TR interaction with coactivators, and prevents full-length wild-type TRβ from achieving transcriptional activation above basal level in this chimeric receptor system.

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