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.[1]References
- Identification in vivo of different rate-limiting steps associated with transcriptional activators in the presence and absence of a GAGA element. Wang, Y.V., Tang, H., Gilmour, D.S. Mol. Cell. Biol. (2005) [Pubmed]
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