title: Identification in vivo of different rate-limiting steps associated with transcriptional activators in the presence and absence of a GAGA element.
abstract: We analyzed the impact of a GAGA element on a transgenic promoter in Drosophila melanogaster that was activated by proteins composed of the Tet(on) DNA binding domain and either the heat shock factor (HSF) activation domain or a potent subdomain of VP16. Permanganate footprinting was used to monitor polymerase II (Pol II) on the transgenic promoters in vivo. Activation by Tet(on)-HSF but not by Tet(on)-VP16(A2) required the GAGA element; this correlated with the ability of the GAGA element to establish a paused Pol II. Although the GAGA element was not required for activation by Tet(on)-VP16(A2), the GAGA element greatly accelerated the rate of activation. The permanganate data also provided evidence that Pol II encountered different rate-limiting steps, following initiation in the presence of Tet(on)-HSF and Tet(on)-VP16(A2). The rate-limiting step in the presence of Tet(on)-HSF was release of Pol II paused about 20 to 40 nucleotides downstream from the start site. The rate-limiting step in the presence of Tet(on)-VP16(A2) occurred much closer to the transcription start site. Several biochemical studies have provided evidence for a structural transition shortly after Pol II initiates transcription. The behavior of Pol II in the presence of Tet(on)-VP16(A2) provides the first evidence that this transition occurs in vivo.
authors.affiliation: Center for Gene Regulation, Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA 16802, USA.
authors.forename: Yunyuan Vivian
authors.initials: YV
authors.lastname: Wang
authors.forename: Hongbing
authors.initials: H
authors.lastname: Tang
authors.forename: David S
authors.initials: DS
authors.lastname: Gilmour
mesh_terms: D000818:Animals; D018983:DNA Footprinting; D004268:DNA-Binding Proteins / Q000235:genetics / Q000502:physiology; D004331:Drosophila melanogaster / Q000235:genetics* / Q000502:physiology; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics*; D006360:Heat-Shock Proteins / Q000235:genetics / Q000502:physiology; D017405:Herpes Simplex Virus Protein Vmw65 / Q000235:genetics / Q000502:physiology; D017434:Protein Structure, Tertiary; D012319:RNA Polymerase II / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000502:physiology; D020218:Response Elements; D015534:Trans-Activators / Q000235:genetics / Q000502:physiology*; D024363:Transcription Initiation Site / Q000502:physiology; D015533:Transcriptional Activation / Q000235:genetics / Q000502:physiology*; D019076:Transgenes
chemical_list: D004268:DNA-Binding Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D017405:Herpes Simplex Virus Protein Vmw65; D011993:Recombinant Fusion Proteins; D015534:Trans-Activators; D012319:RNA Polymerase II