ADENOVIRUS E1B 55K REPRESSES P53 ACTIVATION IN-VITRO

Adenovirus E1B 55K protein cooperates with E1A gene products to induce cell transformation, E1B 55K mediates its effects by binding to and i nhibiting the transcriptional activation and growth suppression functi ons of the tumor suppressor p53. Previous studies in vivo have suggest ed that E1B 55K has an active role in repressing p53 transcriptional a ctivation and that this repression function is directed to specific pr omoters through E1B 55K's interaction with DNA-bound p53. Flag-tagged E1B 55k (e55K) Has expressed with the baculovirus expression system an d immunopurified. Gel filtration, velocity sedimentation centrifugatio n, and glutaraldehyde cross-linking indicated that e55K is a dimer wit h a nonglobular conformation, e55K hound directly to purified p53, cau sing an similar to 10-fold increase in p53 affinity for tandem binding sites, Using in vitro transcription assays reconstituted with purifie d p53, e55K, and HeLa cell nuclear extracts, we found that e55K specif ically repressed p53 activation, These results demonstrate that as pos tulated from earlier transient expression experiments, E1B 55K is a sp ecific repressor of transcription from a promoter with bound p53. Sinc e HeLa nuclear extracts contain little detectable histone protein, E1B 55K probably represses transcription through direct or indirect inter actions with the RNA polymerase II transcription machinery.