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Adenovirus E1B 55K represses p53 activation in vitro
1Molecular Biology Institute, Department of Microbiology and Molecular Genetics, University of California at Los Angeles, 90095-1570, USA.
Abstract:
Adenovirus E1B 55K protein cooperates with E1A gene products to induce cell transformation. E1B 55K mediates its effects by binding to and inhibiting the transcriptional activation and growth-suppression functions of the tumor suppressor p53. Previous studies in vivo have suggested that E1B 55K has an active role in repressing p53 transcriptional activation and that this repression function is directed to specific promoters through E1B 55K's interaction with DNA-bound p53. Flag-tagged E1B 55K (e55K) was expressed with the baculovirus expression system and immunopurified. Gel filtration, velocity sedimentation centrifugation, and glutaraldehyde cross-linking indicated that e55K is a dimer with a nonglobular conformation. e55K bound directly to purified p53, causing an approximately 10-fold increase in p53 affinity for tandem binding sites. Using in vitro transcription assays reconstituted with purified p53, e55K, and HeLa cell nuclear extracts, we found that e55K specifically repressed p53 activation. These results demonstrate that as postulated from earlier transient expression experiments, E1B 55K is a specific repressor of transcription from a promoter with bound p53. Since HeLa nuclear extracts contain little detectable histone protein, E1B 55K probably represses transcription through direct or indirect interactions with the RNA polymerase II transcription machinery.
Insights
Adenovirus E1B 55K protein directly binds and represses the tumor suppressor p53
Area of Science:
- Molecular Biology
- Virology
- Cancer Research
Background:
- Adenovirus E1B 55K protein is crucial for cell transformation.
- E1B 55K inhibits the tumor suppressor p53's functions.
- Previous in vivo studies suggested E1B 55K represses p53 transcriptional activity.
Purpose of the Study:
- To investigate the direct interaction between E1B 55K and p53.
- To elucidate the mechanism by which E1B 55K represses p53 transcriptional activation.
- To determine if E1B 55K directly inhibits p53's function on specific promoters.
Main Methods:
- Expressed and immunopurified flag-tagged E1B 55K (e55K) using a baculovirus system.
- Characterized e55K's physical properties (dimerization, conformation) using gel filtration, velocity sedimentation, and cross-linking.
- Assessed direct binding of e55K to p53 and its effect on p53-DNA binding affinity.
- Utilized in vitro transcription assays with purified components and HeLa nuclear extracts to measure e55K's effect on p53-mediated transcription.
Main Results:
- e55K exists as a dimer with a nonglobular conformation.
- e55K directly binds to p53, increasing p53's affinity for DNA binding sites.
- e55K specifically repressed p53-mediated transcriptional activation in vitro.
- The repression mechanism likely involves interaction with the RNA polymerase II transcription machinery.
Conclusions:
- Adenovirus E1B 55K protein directly represses the transcriptional activation function of tumor suppressor p53.
- E1B 55K's repression is promoter-specific and mediated through interaction with DNA-bound p53.
- This repression likely occurs via direct or indirect interactions with the RNA polymerase II transcription machinery.