Identification in vivo of different rate-limiting steps associated with transcriptional activators in the presence and absence of a GAGA element.
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.
Wang
Yunyuan Vivian
YV
Center for Gene Regulation, Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA 16802, USA.
Tang
Hongbing
H
Gilmour
David S
DS
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
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