title: The ZEBRA activation domain: modular organization and mechanism of action.
abstract: An RNA polymerase II activator often contains several regions that contribute to its potency, an organization ostensibly analogous to the modular architecture of promoters and enhancers. The regulatory significance of this parallel organization has not been systematically explored. We considered this problem by examining the activation domain of the Epstein-Barr virus transactivator ZEBRA. We performed our experiments in vitro so that the activator concentrations, stabilities, and affinities for DNA could be monitored. ZEBRA and various amino-terminal deletion derivatives, expressed in and purified from Escherichia coli, were assayed in a HeLa cell nuclear extract for the ability to activate model reporter templates bearing one, three, five, and seven upstream ZEBRA binding sites. Our data show that ZEBRA contains four modules that contribute to its potency in vitro. The modules operate interchangeably with promoter sites to determine the transcriptional response such that the loss of modules can be compensated for by increasing promoter sites. Potassium permanganate footprinting was used to show that transcriptional stimulation is a consequence of the activator's ability to promote preinitiation complex assembly. Kinetic measurements of transcription complex assembly in a reconstituted system indicate that ZEBRA promotes formation of a subcomplex requiring the TFIIA and TFIID fractions, where TFIIA acts as an antirepressor. We propose a model in which the concentration of DNA-bound activation modules in the vicinity of the gene initiates synergistic transcription complex assembly.
authors.affiliation: Department of Biological Chemistry, UCLA School of Medicine 90024-1737.
authors.forename: T
authors.initials: T
authors.lastname: Chi
authors.forename: M
authors.initials: M
authors.lastname: Carey
mesh_terms: D002467:Cell Nucleus / Q000378:metabolism; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004742:Enhancer Elements, Genetic; D004926:Escherichia coli; D015967:Gene Expression Regulation, Viral; D006367:HeLa Cells; D004854:Herpesvirus 4, Human / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D011401:Promoter Regions, Genetic; D015183:Restriction Mapping; D017384:Sequence Deletion; D016385:TATA Box; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D035561:Transcription Factor TFIIA; D035362:Transcription Factor TFIID; D014157:Transcription Factors / Q000302:isolation & purification / Q000378:metabolism; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000378:metabolism
chemical_list: C063676:BZLF1 protein, Herpesvirus 4, Human; D004268:DNA-Binding Proteins; D015534:Trans-Activators; D035561:Transcription Factor TFIIA; D035362:Transcription Factor TFIID; D014157:Transcription Factors; D014764:Viral Proteins