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Protein/DNA crosslinking of a TFIID complex reveals novel interactions downstream of the transcription start
1Department of Molecular and Cell Biology, Pennsylvania State University, University Park 16802.
Abstract:
A protein--DNA complex containing TFIID has been analyzed by crosslinking. The TBP subunit of TFIID crosslinked to the TATA element but not to any of the regions further downstream which were tested. A 150 kd polypeptide, which corresponds in size to one of the TBP-associated factors (TAFs), crosslinked to a region between +10 and +15 and a second region between +35 and +47. Another polypeptide of greater than 205 kd (also a potential TAF) crosslinked preferentially to the region between +35 and +42. The +10 to +15 region has been recently implicated in hsp70 promoter recognition by TFIID, and the most downstream contacts overlap with the region where RNA polymerase II pauses on the hsp70 promoter in noninduced cells. Crosslinking revealed that as the salt concentration was increased, the TBP interaction was largely unaffected whereas the protein/DNA interactions downstream of the TATA element were disrupted. We propose that during the formation of a transcription complex, TATA-dependent interactions could be disrupted in the vicinity of the start site and the region immediately downstream. A protein contact downstream of +35 might function in pausing polymerase.
Insights
This study used crosslinking to map protein-DNA interactions within the TFIID complex. TFIID binds the TATA element, with TBP interacting there, while TAFs contact downstream regions crucial for transcription regulation.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- Transcription initiation is a complex process involving multiple protein factors.
- TFIID, a key transcription factor, recognizes promoter elements like the TATA box.
- Understanding TFIID's precise interactions with DNA is crucial for deciphering gene regulation.
Purpose of the Study:
- To investigate the specific DNA binding sites of TFIID subunits and associated factors.
- To elucidate the role of TFIID-DNA interactions in promoter recognition and transcription regulation.
- To examine how salt concentration affects these protein-DNA interactions.
Main Methods:
- Chromatin immunoprecipitation (ChIP) followed by crosslinking.
- Analysis of protein-DNA crosslinking patterns using specific antibodies.
- Assessing the impact of varying salt concentrations on protein-DNA complex stability.
Main Results:
- The TATA-binding protein (TBP) subunit of TFIID specifically crosslinked to the TATA element.
- TBP-associated factors (TAFs) crosslinked to downstream promoter regions (+10 to +15 and +35 to +47).
- Downstream interactions were sensitive to salt concentration, unlike the TBP-TATA interaction.
Conclusions:
- TFIID utilizes distinct DNA binding sites for its TBP subunit and TAFs.
- Downstream TFIID-DNA contacts may be transient or involved in regulating transcription start site selection and polymerase pausing.
- TFIID's interaction dynamics at the promoter are sensitive to ionic strength, suggesting conformational changes during transcription initiation.