title: Transcriptional synergy by the Epstein-Barr virus transactivator ZEBRA.
abstract: ZEBRA is an Epstein-Barr virus (EBV) transcriptional activator that mediates a genetic switch between the latent and lytic states of the virus by binding to the promoters of genes involved in lytic DNA replication and activating their transcription. A computer survey revealed that 9 of 23 potential or known ZEBRA-responsive EBV genes contained two or more upstream binding sites; this suggested that ZEBRA can stimulate transcription synergistically. By using a series of synthetic promoters bearing one, two, three, five, and seven upstream recognition sites, we showed that ZEBRA activates transcription synergistically when templates bearing multiple sites were compared with a template bearing a single site. This phenomenon was observed in both uninfected and EBV-infected B-lymphoid cells and in vitro in a HeLa cell nuclear extract. DNase I footprinting was used to show that the synergy was not due to cooperative DNA binding mediated by direct contact between ZEBRA dimers. The in vitro experiments revealed two manifestations of synergy. One was seen when the levels of transcription observed with the same amounts of ZEBRA added to templates bearing different numbers of sites were compared. The other was observed when the two lowest concentrations of ZEBRA that stimulated measurable transcription from any given template were compared. On the basis of both the number of sites and the calculated Kd of ZEBRA for a single site, we estimated that the critical concentration of ZEBRA needed to elicit transcriptional synergy corresponds to a site occupancy of two or three bound ZEBRA dimers. Our results have biologic implications for both the EBV lytic cycle and other processes in which the concentration of an activator changes either temporally or spatially.
authors.affiliation: Department of Biological Chemistry, UCLA School of Medicine 90024-1737.
authors.forename: M
authors.initials: M
authors.lastname: Carey
authors.forename: J
authors.initials: J
authors.lastname: Kolman
authors.forename: D A
authors.initials: DA
authors.lastname: Katz
authors.forename: L
authors.initials: L
authors.lastname: Gradoville
authors.forename: L
authors.initials: L
authors.lastname: Barberis
authors.forename: G
authors.initials: G
authors.lastname: Miller
mesh_terms: D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D002467:Cell Nucleus / Q000502:physiology; D003001:Cloning, Molecular; D004279:DNA, Viral / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D003850:Deoxyribonuclease I; D004926:Escherichia coli / Q000235:genetics; D015967:Gene Expression Regulation, Viral; D005814:Genes, Viral; D006367:HeLa Cells; D004854:Herpesvirus 4, Human / Q000235:genetics*; D006801:Humans; D007700:Kinetics; D008957:Models, Genetic; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D016385:TATA Box; D013698:Templates, Genetic; D015534:Trans-Activators / Q000378:metabolism*; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014764:Viral Proteins
chemical_list: C063676:BZLF1 protein, Herpesvirus 4, Human; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; D015534:Trans-Activators; D014764:Viral Proteins; D003850:Deoxyribonuclease I