Open AccessIdentification In Vivo of Different Rate-Limiting Steps Associated with Transcriptional Activators in the Presence and Absence of a GAGA Element
Author(s) -
Yunyuan Vivian Wang,
Hongbing Tang,
David S. Gilmour
Publication year - 2005
Publication title -
molecular and cellular biology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.14
H-Index - 327
eISSN - 1067-8824
pISSN - 0270-7306
DOI - 10.1128/mcb.25.9.3543-3552.2005
Subject(s) - biology , footprinting , transcription (linguistics) , microbiology and biotechnology , transcription factor , rna polymerase ii , promoter , biochemistry , gene , gene expression , linguistics , philosophy
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.