Quantitative FRET imaging in living cells is made possible on a simple fluorescence microscope with this new method. The single-step calibration is robust and gives absolute FRET values, independent of the instrument.
Gene expression is highly controlled and regulated in living cells. One of the first steps in gene transcription is
recognition of the promoter site by the TATA box Binding Protein (TBP). TBP recruits other transcriptions factors and
eventually the RNA polymerase II to transcribe the DNA in mRNA. We developed a single pair Förster Resonance
Energy Transfer (spFRET) assay to investigate the mechanism of gene regulation. Here, we apply this assay to
investigate the initial binding process of TBP to the adenovirus major late (AdML) promoter site. From the spFRET
measurements, we were able to identify two conformations of the TBP-DNA complex that correspond to TBP bound in
the correct and the opposite orientation. Increased incubation times or the presence of the transcription factor TFIIA
improved the alignment of TBP on the promoter site. Binding of TBP to the TATA box shows a rich dynamics with
abrupt transitions between multiple FRET states. A frame-wise histogram analysis revealed the presence of at least six
discrete states, showing that TBP binding is more complicated than previously thought. Hence, the spFRET assay is very
sensitive to the conformation of the TBP-DNA complex and is very promising tool for investigating the pathway of TBP
binding in detail.
Access to the requested content is limited to institutions that have purchased or subscribe to SPIE eBooks.
You are receiving this notice because your organization may not have SPIE eBooks access.*
*Shibboleth/Open Athens users─please
sign in
to access your institution's subscriptions.
To obtain this item, you may purchase the complete book in print or electronic format on
SPIE.org.
INSTITUTIONAL Select your institution to access the SPIE Digital Library.
PERSONAL Sign in with your SPIE account to access your personal subscriptions or to use specific features such as save to my library, sign up for alerts, save searches, etc.