DNA binding site selection by RNA polymerase II TAFs: a TAF(II)250-TAF(II)150 complex recognizes the initiator

Area of Science:

• Molecular Biology
• Gene Regulation
• Biochemistry

Background:

• Basal transcription factor TFIID is crucial for RNA polymerase II transcription.
• TFIID is composed of the TATA-box-binding protein (TBP) and TBP-associated factors (TAFs).
• TAFII250 and TAFII150 have been previously implicated in core promoter selectivity.

Purpose of the Study:

• To identify the specific DNA sequences recognized by TAFII250 and TAFII150.
• To investigate the role of TAFIIs in promoter recognition and basal transcription.
• To understand how TAFII binding influences promoter responsiveness to transcriptional activators.

Main Methods:

• Random DNA-binding site selection to identify TAF target sequences.
• In vitro transcription assays using reconstituted basal transcription systems.
• Site-directed mutagenesis to probe the functional importance of TAF-DNA interactions.

Main Results:

• Neither TAFII250 nor TAFII150 alone binds a constrained DNA sequence.
• A TAFII250-TAFII150 complex specifically selects sequences matching the Initiator (Inr) consensus.
• The TAFII250-TAFII150 complex, when associated with TBP, recognizes Inr sequences at a precise distance from the TATA-box.
• Mutations disrupting TAFII-Inr binding impair Inr function in transcription.
• TAFII150 also binds to four-way junction DNA, suggesting structural DNA recognition by TFIID.
• The DNA sequence at the transcription start site affects Sp1-mediated transcriptional regulation.

Conclusions:

• TAFII250 and TAFII150 directly bind the Inr sequence.
• Inr recognition by TFIID is functionally relevant for basal transcription.
• Inr recognition by TFIID can dictate promoter responsiveness to activators.
• TFIID may recognize both DNA sequence and structure for promoter binding.