Trans activation by the bovine papillomavirus E2 protein in Saccharomyces cerevisiae

Morrissey, L C; Barsoum, James; Androphy, Elliot J.

doi:10.1128/jvi.63.10.4422-4425.1989

Cited by 37 publications

(23 citation statements)

References 34 publications

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“…from a promoter with two mutated E2 binding sites as the upstream element. It has been previously reported that amino-terminally truncated E2 proteins such as E2-R do not activate the E2 enhancer motif in rodent (Lambert et al, 1987;Haugen et al, 1988) and yeast cells (Morrissey et al, 1989). Since PV infection and pathologic effects are generally limited to epithelial cells (an exception is fibroma in some animals) we questioned whether E6 trans-activation function was operative in the lower eukaryote S. cerevisiae.…”

Section: Resultsmentioning

confidence: 95%

“…Since a BPV E6 responsive DNA element has not been identified, the E6 coding region was molecularly cloned onto the DNA binding domain of BPV E2, creating E6-E2-R ( Figure 1). This E2-R region lacked the E2 activation domain and is unable to induce E2 elements in mammalian cells and yeast (Lambert et al, 1989;Morrissey et al, 1989). The fusion gene was constructed to preserve the BPV E6 protein, with the only alteration of E6 limited to a conservative change of lysine to arginine two amino acids prior to its terminal proline.…”

Section: Resultsmentioning

confidence: 99%

“…When E6 alone (pYE6) was introduced into yeast strain BGW1-7a along with the lacZ gene that carried one, two or four copies of the E2 binding site (pBY-1, 2 or 4, respectively; Morrissey et al, 1989), no significant increase in the expression of $-gal over levels with the vector pKP-15 were detected with cells cultured in galactose (Table II). The series of E6-E2 DNA binding domain fusion constructions were then co-transformed along with the reported promoter plasmids and colonies that contained both constructions were selected on media deficient for uracil and leucine.…”

Section: Resultsmentioning

confidence: 99%

“…The LEU based yeast target plasmids pBY-1, -2 and -4 that contain one, two or four E2 binding sites upstream from the CYC-1 TATA elements and the lacZ gene were transformed into S.cerevisiae strain BGWI-7a (leuura-) (Morrissey et al, 1989). LEU+ URA+ colonies were initially amplified in glucose, and switched to galactose containing media 12 h prior to assay.…”

Section: Discussionmentioning

confidence: 99%

“…This restored BamHI ends and retained the first ATG codon in the reading frame. E6 and the E6-E2-R fragments from the pUC vector described below were subsequently cloned into the BamHI site of the pZipNeoSV(X)-1 vector (kindly provided by R.Cone) or the URA based yeast expression vector pKP15 which included the GAL UAS and CYC-1 promoter, a high copy number 2M replication origin and URA gene (Morrissey et al, 1989).…”

Section: Constructionsmentioning

confidence: 99%

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Transcriptional activation by the papillomavirus E6 zinc finger oncoprotein.

et al. 1990

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The introduction of the bovine (BPV) or human papillomavirus E6 gene into susceptible cells can result in their transformation, but there are few clues to the mechanism of action of the E6 gene. The characteristic features of E6 proteins are their small size (approximately 150 amino acids) and the potential to form two large zinc fingers. To determine if E6 can function as a transcription factor, the BPV E6 gene was fused to the sequence specific DNA binding peptide encoded by the BPV E2 gene. This chimeric E6‐E2 protein trans‐activated promoters that incorporated E2 binding elements in both rodent cells and Saccharomyces cerevisiae. In the absence of E6‐E2 localization to the target promoter, trans‐activation did not occur. Alteration of the cysteine residues at the base of each finger abrogated the transcriptional activity of the E6‐E2 hybrids. These data demonstrated that the BPV E6 gene encodes a transcription activation domain and imply that a specific structure of the protein, most likely the zinc fingers, is critical for this function. Since these cysteine mutants are also transformation defective, E6 transcriptional functions may be required for its oncogenic activity.

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Section: Resultsmentioning

confidence: 95%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Constructionsmentioning

confidence: 99%

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Transcriptional activation by the papillomavirus E6 zinc finger oncoprotein.

et al. 1990

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Several different upstream promoter elements can potentiate transactivation by the BPV-1 E2 protein.

et al. 1991

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The enhancer and upstream promoter regions of RNA polymerase II transcribed genes modulate the rate of transcription initiation and establish specific patterns of gene expression. Both types of region consist of clusters of DNA binding sites for nuclear proteins. To determine how efficiently the same factor can activate transcription when acting as an enhancer or promoter factor, we have studied transactivation by the BPV‐1 E2 protein, a papillomavirus transcriptional regulator. By cotransfecting a BPV‐1 E2 expression vector and a series of reporter plasmids containing well‐defined chimeric promoters we have found that whilst E2 can strongly stimulate complex promoters such as that of the HSV tk gene, it does not efficiently activate constructions containing only a TATA box and initiation site. We show that insertion of upstream promoter elements, but not of spacer DNA, between E2 binding sites and the TATA box greatly increases E2 activation. This effect was observed with more than one type of upstream promoter element, is not related to the strength of the promoter and is unlikely to result from co‐operative DNA binding by E2 and the transcription factors tested. These results would suggest that E2 has the properties of an enhancer rather than promoter factor and that in certain cases promoter and enhancer factors may affect different steps in the process of transcriptional activation.

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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.

et al. 1995

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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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Trans activation by the bovine papillomavirus E2 protein in Saccharomyces cerevisiae

Cited by 37 publications

References 34 publications

Transcriptional activation by the papillomavirus E6 zinc finger oncoprotein.

Transcriptional activation by the papillomavirus E6 zinc finger oncoprotein.

Several different upstream promoter elements can potentiate transactivation by the BPV-1 E2 protein.

The bovine papillomavirus 1 E2 protein contains two activation domains: one that interacts with TBP and another that functions after TBP binding.

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