Identification in vivo of different rate-limiting steps associated with transcriptional activators in the presence and absence of a GAGA element

Yunyuan Vivian Wang, Hongbing Tang, David S. Gilmour

Research output: Contribution to journalArticlepeer-review

20 Scopus citations

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 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.

Original languageEnglish (US)
Pages (from-to)3543-3552
Number of pages10
JournalMolecular and cellular biology
Volume25
Issue number9
DOIs
StatePublished - May 2005

All Science Journal Classification (ASJC) codes

  • Molecular Biology
  • Cell Biology

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