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 Teton-HSF but not by Teton-VP16A2 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 Teton-VP16A2, 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 Teton-HSF and Teton-VP16A2. The rate-limiting step in the presence of Teton-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 Teton-VP16A2 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-VP16A2 provides the first evidence that this transition occurs in vivo.
All Science Journal Classification (ASJC) codes
- Molecular Biology
- Cell Biology