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Moshe Yaniv - One of the best experts on this subject based on the ideXlab platform.
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the Bovine Papillomavirus 1 e2 protein contains two activation domains one that interacts with tbp and another that functions after tbp binding
The EMBO Journal, 1995Co-Authors: Gertrud Steger, J Ham, O Lefebvre, Moshe YanivAbstract:The E2 transactivator of Bovine Papillomavirus type-1 is unable to activate minimal promoters in vivo that contain only E2 binding sites and a TATA box. This block can be overcome by over-expression of human TATA binding protein (TBP) or by the addition of either SP1 binding sites or an initiator element to the promoter, suggesting that the binding of TFIID may normally be a rate-limiting step for activation by E2. Surprisingly, purified E2 and TBP bind co-operatively to DNA in vitro when the sites are closely spaced. E2 does not affect the on rate of association but reduces the off rate. The E2 region responsible for this effect is located in the hinge region that links the classic transactivation and DNA binding domains. We demonstrate that the TBP stabilizing domain contributes in vivo to co-operativity with co-expressed TBP and to activation of the major late minimal promoter (MLP) containing E2 sites. In contrast, promoters with SP1 sites are activated to wild-type levels by such a mutant. This promoter specificity is also evident in vitro. A truncated E2 mutant, lacking the classic transactivation domain but containing the TBP stabilizing domain, stimulates transcription of the MLP in vitro, but does not activate promoters with SP1 sites. In conclusion, our results show that the E2 transactivation domain has a modular structure. We have identified one domain which probably acts at an early step in the assembly of the pre-initiation complex and which is involved in reducing the dissociation rate of bound TBP in vitro. The classic N-terminal activation domain of E2 might affect one or several step(s) in the assembly of the preinitiation complex occurring after the binding of TFIID.
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cooperativity in vivo between the e2 transactivator and the tata box binding protein depends on core promoter structure
The EMBO Journal, 1994Co-Authors: Gerhard Steger, Moshe YanivAbstract:The E2 transactivator protein of Bovine Papillomavirus 1 (BPV-1) can strongly stimulate complex promoters such as that of the herpes simplex virus thymidine kinase gene but does not efficiently activate minimal promoters that only contain E2 binding sites and a TATA box. Here we show that overexpression of the human, but not yeast, TATA box binding protein (TBP) in transfection experiments overcomes this block and enables E2 to activate a minimal TATA box-containing promoter. This suggests that recruitment of the TFIID complex to such promoters is normally a rate limiting step for transcriptional activation by E2 in vivo. In contrast, minimal promoters that contain an initiator element in addition to a TATA box are efficiently activated by E2 on its own and this activation is only moderately enhanced by TBP overexpression. In such E2-responsive promoters the TATA box or initiator can be functionally replaced by SP1 binding sites. Both the initiator binding protein, TFII-I, and SP1 have been found to interact physically with components of the TFIID complex. Since either TBP overexpression or the presence of an initiator or SP1 binding sites can increase activation by E2, it seems likely that the principal role of the E2 activation domain is to affect a step in the formation of the transcription initiation complex that occurs after TFIID has bound to the promoter. Sequential action of transcription factors, such as TFII-I, SP1 and E2, may be one type of mechanism underlying the widely observed phenomenon of transcriptional synergy.
Rashmi S Hegde - One of the best experts on this subject based on the ideXlab platform.
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subunit rearrangement accompanies sequence specific dna binding by the Bovine Papillomavirus 1 e2 protein
Journal of Molecular Biology, 1998Co-Authors: Rashmi S Hegde, Aifei Wang, Seungsup Kim, Matthieu SchapiraAbstract:The 2.5 A crystal structures of the DNA-binding domain of the E2 protein from Bovine Papillomavirus strain 1 and its complex with DNA are presented. E2 is a transcriptional regulatory protein that is also involved in viral DNA replication. It is the structural prototype for a novel class of DNA-binding proteins: dimeric beta-barrels with surface alpha-helices that serve as recognition helices. These helices contain the amino-acid residues involved in sequence-specifying interactions. The E2 proteins from different Papillomavirus strains recognize and bind to the same consensus 12 base-pair DNA sequence. However, recent evidence from solution studies points to differences in the mechanisms by which E2 from the related viral strains Bovine Papillomavirus-1 and human Papillomavirus-16 discriminate between DNA targets based on non-contacted nucleotide sequences. This report provides evidence that sequence-specific DNA-binding is accompanied by a rearrangement of protein subunits and deformation of the DNA. These results suggest that, along with DNA sequence-dependent conformational properties, protein subunit orientation plays a significant role in the mechanisms of target selection utilized by E2.
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crystal structure at 1 7 a of the Bovine Papillomavirus 1 e2 dna binding domain bound to its dna target
Nature, 1992Co-Authors: Rashmi S Hegde, Steven R Grossman, Laimonis A Laimins, Paul B SiglerAbstract:The dominant transcriptional regulator of the papiIlomaviruses, E2, binds to its specific DNA target through a previously unobserved dimeric ant i para I lei β-barrel. The DNA is severely but smoothly bent over the barrel by the interaction of successive major grooves with a pair of symmetrically disposed α-helices. The specific interface is an 'interwoven' network of interactions where the identifying base pairs of the target contact more than one amino-acid side chain and the discriminating amino acids interact with more than one base pair.
Andres Metspalu - One of the best experts on this subject based on the ideXlab platform.
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a Bovine Papillomavirus 1 based vector restores the function of the low density lipoprotein receptor in the receptor deficient cho ldla7 cell line
BMC Molecular Biology, 2002Co-Authors: Jaana Tammur, Mart Ustav, Hiljar Sibul, Ene Ustav, Andres MetspaluAbstract:The rationale of using Bovine Papillomavirus-1 (BPV-1) derived vectors in gene therapy protocols lies in their episomal maintenance at intermediate to high copy number, and stable, high-level expression of the gene products. We constructed the BPV-1 based vector harbouring the human low-density lipoprotein receptor (LDLR) gene cDNA and tested its ability to restore the function of the LDLR in the receptor-deficient cell line CHO-ldlA7. The introduced vector p3.7LDL produced functionally active LDL receptors in the receptor-deficient cell line CHO-ldlA7 during the 32-week period of observation as determined by the internalisation assay with the labelled LDL particles. Bovine Papillomavirus type-1 (BPV-1)-derived vectors could be suitable for gene therapy due to their episomal maintenance at intermediate to high copy number and stable, high-level expression of the gene products. The constructed BPV-1 based vector p3.7LDL produced functionally active LDL receptors in the LDLR-deficient cell line CHO-ldlA7 during the 32-week period of observation. In vivo experiments should reveal, whether 1–5% transfection efficiency obtained in the current work is sufficient to bring about detectable and clinically significant lowering of the amount of circulating LDL cholesterol particles.
Scott Vande B Pol - One of the best experts on this subject based on the ideXlab platform.
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interactions between e6 fak and git1 at paxillin ld4 are necessary for transformation by Bovine Papillomavirus 1 e6
Journal of Virology, 2014Co-Authors: Nicole Brimer, Ramon Wade, Scott Vande B PolAbstract:Bovine Papillomavirus 1 E6 interacts with two similar proteins that regulate cell attachment and cell migration called paxillin (PXN) and HIC-5 (also known as HIC5, ARA55, HIC-5, TSC-5, and TGFB1I1). Despite the similarity between HIC-5 and paxillin, paxillin is required for E6 to transform mouse embryo fibroblasts while HIC-5 is not. Using mutants of paxillin, we found that dynamic competitive interactions between E6, focal adhesion kinase, and the GIT1 ARF-GAP protein for binding to paxillin are required but not sufficient for transformation by E6. Using mutants of paxillin and chimeric proteins between HIC-5 and paxillin, we demonstrate that a critical difference between HIC-5 and paxillin is within the LIM domains of paxillin that do not directly interact with E6. Mutational analysis indicates that at least six distinct domains of paxillin are required for E6 transformation. IMPORTANCE Papillomaviruses cause epitheliomas in vertebrates through the actions of virus-encoded oncoproteins. Despite the immense diversity of Papillomavirus types, our understanding of the mechanisms by which the virus-encoded E6 oncoproteins contribute to cell transformation is restricted to human Papillomavirus types that are associated with cancer. Bovine Papillomavirus 1 (BPV-1) E6 has served as a model system for studies of E6 structure and function. This study examines the mechanisms by which BPV-1 E6 association with the cellular focal adhesion adapter protein paxillin contributes to cell transformation and extends our knowledge of the diverse mechanisms by which Papillomaviruses transform host cells.
Alison A Mcbride - One of the best experts on this subject based on the ideXlab platform.
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separation of the transcriptional activation and replication functions of the Bovine Papillomavirus 1 e2 protein
The EMBO Journal, 1992Co-Authors: Patricia L Winokur, Alison A McbrideAbstract:Replication of Bovine Papillomavirus-1 (BPV-1) DNA requires two viral gene products, the E1 protein and the full-length E2 protein. The 48 kDa E2 protein is a site-specific DNA-binding protein that binds to several sites which lie adjacent to the BPV-1 origin of replication. The 85 amino acid C-terminal domain contains the specific DNA binding and dimerization properties of the protein. The approximately 200 amino acid N-terminal domain is crucial for transcriptional activation. Both of these domains are highly conserved among different Papillomaviruses. An internal hinge region separates the two functional domains. The region varies in amino acid sequence and length among the E2 proteins of different Papillomaviruses. A series of mutations were constructed within the E2 open reading frame which delete various regions of the conserved DNA binding and transactivation domains as well as the internal hinge region. Two mutated E2 proteins that lack portions of the conserved DNA-binding domain but which support DNA replication were identified using transient replication assays. These mutated E2 proteins were unable to function as transcriptional activators. Conversely, two E2 proteins containing large deletions of the hinge region were able to activate transcription, but were defective for replication. Thus, the replication and transactivation functions of the E2 protein are separable.