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Molecular and Cellular Biology, May 2005, p. 3543-3552, Vol. 25, No. 9
0270-7306/05/$08.00+0     doi:10.1128/MCB.25.9.3543-3552.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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, and David S. Gilmour*

Center for Gene Regulation, Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania

Received 9 January 2005/ Accepted 19 January 2005

We analyzed the impact of a GAGA element on a transgenic promoter in Drosophila melanogaster that was activated by proteins composed of the Teton 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 Teton-VP16A2 provides the first evidence that this transition occurs in vivo.

* Corresponding author. Mailing address: Center for Gene Regulation, Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA 16802. Phone: (814) 863-8905. Fax: (814) 863-7024. E-mail: dsg11{at}psu.edu.

Molecular and Cellular Biology, May 2005, p. 3543-3552, Vol. 25, No. 9
0022-538X/05/$08.00+0     doi:10.1128/MCB.25.9.3543-3552.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Hendrix, D. A., Hong, J.-W., Zeitlinger, J., Rokhsar, D. S., Levine, M. S. (2008). Promoter elements associated with RNA Pol II stalling in the Drosophila embryo. Proc. Natl. Acad. Sci. USA 105: 7762-7767 [Abstract] [Full Text]  
  • Lee, C., Li, X., Hechmer, A., Eisen, M., Biggin, M. D., Venters, B. J., Jiang, C., Li, J., Pugh, B. F., Gilmour, D. S. (2008). NELF and GAGA Factor Are Linked to Promoter-Proximal Pausing at Many Genes in Drosophila. Mol. Cell. Biol. 28: 3290-3300 [Abstract] [Full Text]  
  • Core, L. J., Lis, J. T. (2008). Transcription Regulation Through Promoter-Proximal Pausing of RNA Polymerase II. Science 319: 1791-1792 [Abstract] [Full Text]  


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