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.