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Query: UNIPROT:P04637 (p53)
 77,613 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Transcription associated with a terminal deoxynucleotide transferase gene initiator element is shown to respond to the transcription factor GAL4-VP16 both in vivo and in vitro. High-level transcription requires both an intact initiator element and bound activator. Transcription from this initiator-directed promoter is synergistic in vivo in that five GAL4 DNA binding sites yield 36 times the expression of a single site. Promoters dominated by initiator and TATA elements respond similarly to several GAL4-based activators, including GAL4-Sp1, GAL4-CTF, GAL4(1-147), GAL4-p53, GAL4-C/EBP, and GAL4-ER(EF), as well as GAL4-VP16 and Sp1. These and other similarities suggest that primary activation of TATA- and initiator-dominated promoters occurs at common steps. Since the initial assembly steps do not appear to be common for the two promoter types, the results place interesting constraints on models for how activation occurs.
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PMID:Properties of initiator-associated transcription mediated by GAL4-VP16. 824 64

Many DNA tumor viruses express a protein that inhibits transcriptional activation by the tumor-suppressing transcription factor p53. We report that adenovirus E1B 55K represses p53-mediated activation by a mechanism not described previously. E1B 55K binds p53 without displacing it from its DNA-binding site. A fusion of E1B 55K to the GAL4 DNA-binding domain represses transcription from a variety of promoters with engineered upstream GAL4-binding sites. Mutations within E1B 55K that interfere with its transforming activity and its ability to inhibit p53-mediated trans-activation also interfere with transcriptional repression by the GAL4-55K fusion. These results demonstrate that E1B 55K functions as a direct transcriptional repressor that is targeted to p53-responsive genes by binding to p53.
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PMID:Adenovirus E1B oncoprotein tethers a transcriptional repression domain to p53. 829 38

Recent evidence suggests that the tumor-suppressor protein p53 functions as a transcriptional regulator to control cell proliferation. An interaction with p53 is required for SV40 T antigen to transform primary cells; however, the effect of T antigen binding on p53 function is not known. In order to determine if an interaction with T antigen results in loss of p53-mediated transcriptional activity, we have used vectors expressing either a p53-GAL4 fusion protein or a wild-type p53 protein in transient co-transfection assays with T-antigen expression vectors. We have demonstrated that coexpression of T antigen significantly reduces both p53-GAL4-mediated transcription from a GAL4-dependent CAT reporter and p53-mediated transcription from a consensus p53 binding site in vivo. Moreover, T antigen was able to reduce binding of p53-GAL4 to its GAL4 binding sequence in gel shift experiments in vitro. These observed activities of T antigen were all dependent upon a functional p53-binding domain. In addition, coexpression of human papillomavirus type 18 E6 protein, able to bind to p53, was able to significantly reduce p53-mediated transcription. These results suggest that an interaction of certain viral oncoproteins with p53 results in loss of transcriptional activity of p53, a function that is important for maintaining normal cell growth.
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PMID:SV40 T antigen abrogates p53-mediated transcriptional activity. 837 89

For papillomavirus DNA replication, the E2 enhancer protein cooperatively assists in binding of the E1 helicase to the origin. We report that, at limiting E1 and E2 levels, the enhancer proteins GAL4-VP16 and GAL4-p53(1-73) stimulate BPV in vitro DNA replication. This cell-free system was used to ascertain whether the acidic activation domains have a cellular target important for replication. Cellular extracts were depleted of replication activity by passage through a VP16 affinity column. The protein depleted was the cellular factor replication protein A. The direct interaction between replication protein A and VP16, as well as the activation of replication by VP16, is dependent upon the C-terminus of the VP16 activation domain. E2 and the activation domain of p53 also interact with replication protein A. We suggest that a link between transcription and replication involves factors that help convert a closed DNA complex to an open complex.
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PMID:The acidic transcriptional activation domains of VP16 and p53 bind the cellular replication protein A and stimulate in vitro BPV-1 DNA replication. 839 Mar 28

Germline mutations in the tumor-suppressor p53 have been recently identified in Li-Fraumeni syndrome patients. We analysed the function of one of these mutations, an arg-to-trp substitution at amino acid 245 in the murine p53 gene. This p53LFS mutant could not, unlike wild-type p53, suppress foci formation of rat embryo-fibroblasts. Like other p53 mutants it cooperated with activated ras to transform rat embryo fibroblasts. Overexpression of p53LFS thus resulted in a phenotype similar to other mutant p53s. The p53LFS protein was also transcriptionally inactive in contrast to previous studies using a p53LFS/GAL4 fusion protein. To better understand the functional domain disrupted in p53LFS, we developed a dimerization assay and showed that p53LFS still dimerized. In addition, p53LFS retained its ability to bind SV40 large T antigen and not hsc70, both characteristics of wild-type p53. Using immunofluorescence, we localized p53LFS to the nucleus. From these results we conclude that p53LFS represents an unusual p53 mutant in that it retains many characteristics of wild-type p53, however activities critical for growth suppression are lost.
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PMID:A functionally inactive p53 Li-Fraumeni syndrome mutant. 842 39

We used a yeast-based genetic assay, the two-hybrid system, to characterize the domain of the tumor-suppressor p53 involved in oligomerization. This assay relies on the reconstitution of the function of a transcriptional activator, the yeast GAL4 protein, via the interaction of a protein fused to the DNA-binding domain of GAL4 with a protein fused to the transcriptional activation domain of GAL4. We show by a reconstruction experiment that this approach could detect the interaction of p53 deleted for its N-terminal activation domain with SV40 large T antigen. We then searched a library of human proteins present as activation domain hybrids for proteins that can interact with the hybrid of p53 with the DNA-binding domain. This search identified 36 plasmids containing the p53 gene, representing 10 different classes. These results provide an additional in vivo demonstration of p53 oligomerization. The smallest p53 fragment identified from screening the library contained only amino acids 331-393, indicating that this small C-terminal fragment is sufficient to mediate oligomerization. In addition, a mutant p53 protein could bind to the wild-type protein in this assay, providing support for the idea that mutant forms of p53 act in a dominant-negative manner through C-terminal oligomerization with the wild type.
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PMID:Use of the two-hybrid system to identify the domain of p53 involved in oligomerization. 850 89

Accumulating evidence supports the hypothesis that tumor-suppressor p53 can act as a transcriptional activator. Insertion of high-affinity p53 DNA binding sites upstream of a promoter yields a p53-responsive vector. Chimeric proteins fusing p53 and the GAL4 DNA-binding domain demonstrate the presence of a transcriptional activating domain in the N-terminus of p53. GAL4-p53 chimeras constructed using naturally occurring p53 mutations at either codon 141 (Tyr-141) or 175 (His-175) of p53 had little ability to activate the reporter gene; in contrast, mutations at either codon 248 (Trp-248) or 273 (His-273) produced greater transcriptional activities than did wild-type p53. GAL4 chimeras can be used to analyse interactions between different domains of p53 and between different p53 alleles; a DNA binding site is defined, and a simple measurement can be made of function. We had expected that coexpression of GAL4 chimeras and p53 alleles would squelch transcriptional activation downstream of GAL binding sites. Surprisingly, coexpression of either p53 (Trp-248) or (His-273) with the GALA-p53 (wild-type, His-273, Trp-248, His-175, Tyr-141) effectors conferred an increase in transcriptional activation as compared with the effector alone. Oligomerization of p53 alleles with GAL4-p53 chimeras could underlie this effect, leading to an increase in transcription-activating motifs near the promoter. To test this possibility, we constructed a GAL4-p53 C-terminal chimera with p53 residues 160-393, lacking the transcriptional activating domain but retaining regions believed to be important in p53 oligomerization. Neither GAL4-p53 (C-terminus) nor p53 expression vectors were able to transactivate G5E1B-CAT alone. Both p53 (His-273) and (Trp-248) co-expressed with GAL4-p53 (C-terminus) were able to transactivate the G5E1B-CAT reporter gene; in contrast, p53 (Tyr-141) was not able to activate transcription. p53 (Tyr-141/His-273) behaved as a dominant negative mutant and inhibited the ability of the combination of p53 (His-273) and GAL4-p53 (C-terminus) to stimulate the reporter gene. Double immunoprecipitation by sequentially using GAL4 and p53 antibodies showed that p53 (His-273) and (Tyr-141/His-273), but not p53 (Tyr-141), can efficiently oligomerize in vivo to the C-terminal region of p53. Transcriptional activating function of p53 may be modulated by oligomerization; some mutations, such as His-273 and Trp-248, participate in these functions.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Mutant p53 proteins have diverse intracellular abilities to oligomerize and activate transcription. 851 Sep 27

Many transcription factors can activate the initiation of DNA replication. We have used affinity chromatography to show that the acidic activation domains of the transcription factors VP16, GAL4, and p53 each bind selectively to human and yeast replication factor A (RPA). The binding is direct and to the largest subunit of the trimeric RPA complex, RPA-1. Mutations in VP16 that reduce the ability of GAL4-VP16 to activate polyomavirus DNA replication also compromise the binding of VP16 to RPA. We suggest that transcription factors may interact with RPA either to stabilize single-stranded DNA at a replication origin or to recruit DNA polymerase alpha to the replication initiation complex.
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PMID:The transactivator proteins VP16 and GAL4 bind replication factor A. 851 4

The p53 tumor suppressor gene product is a sequence-specific DNA-binding protein that is necessary for the G1 arrest of many cell types. Consistent with its role as a cell cycle checkpoint factor, p53 has been shown to be capable of both transcriptional activation and repression. Here we show a new potential role for p53 as a DNA-binding-dependent regulator of DNA replication. Constructs containing multiple copies of the ribosomal gene cluster (RGC) p53 binding site cloned on the late side of the polyomavirus origin were used in in vitro replication assays. In the presence of p53, the replication of these constructs was strongly inhibited, while the replication of constructs containing a mutant version of the RGC site was not affected by p53. Several tumor-derived mutant p53 proteins were unable to inhibit replication of the construct with wild-type RGC sites. Additionally, the transactivator GAL4-VP16 was unable to inhibit replication of a construct containing GAL4 binding sites adjacent to the polyomavirus origin. We also show that the inhibition by p53 can occur from sites cloned as far as 600 bp from the origin. Preincubation experiments suggest that p53 inhibits replication at a step mediated by ATP, possibly by inhibiting the binding of polyomavirus T antigen to the core origin. The presence of an endogenous p53 binding site in the polyomavirus origin suggests potential mechanisms for the observed inhibition.
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PMID:p53 inhibits DNA replication in vitro in a DNA-binding-dependent manner. 852 20

p53 stimulates the transcription of a number of genes, such as MDM2, Waf1, and GADD45. We and others have shown previously that this activity of p53 can be inhibited by adenovirus type 2 or 12 large E1B proteins. Here we show that the adenovirus E1A proteins also can repress the stimulation of transcription by p53, both in transient transfections and in stably transfected cell lines. The inhibition by E1A occurs without a significant effect on the DNA-binding capacity of p53. Furthermore, the activity of a fusion protein containing the N-terminal part of p53 linked to the GAL4 DNA-binding domain can be suppressed by E1A. This indicates that E1A affects the transcription activation domain of p53, although tryptic phosphopeptide mapping revealed that the level of phosphorylation of this domain does not change significantly in E1A-expressing cell lines. Gel filtration studies, however, showed p53 to be present in complexes of increased molecular weight as a result of E1A expression. Apparently, E1A can cause increased homo- or hetero-oligomerization of p53, which might result in the inactivation of the transcription activation domain of p53. Additionally, we found that transfectants stably expressing E1A have lost the ability to arrest in G1 after DNA damage, indicating that E1A can abolish the normal biological function of p53.
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PMID:Adenovirus E1A proteins inhibit activation of transcription by p53. 862 76


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