Nucleic Acids Research Advance Access published online on September 29, 2008
Nucleic Acids Research, doi:10.1093/nar/gkn612
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Gene regulation, Chromatin and Epigenetics |
Nonradioactive, ultrasensitive site-specific protein–protein photocrosslinking: interactions of 
-helix 2 of TATA-binding protein with general transcription factor TFIIA and transcriptional repressor NC2
1Howard Hughes Medical Institute, Waksman Institute, and Department of Chemistry and Chemical Biology, Rutgers University, Piscataway NJ 08854 and 2Howard Hughes Medical Institute and Department of Biochemistry, New York University Medical School, New York NY 10016, USA
*To whom correspondence should be addressed. Tel: 732 445 5179; Fax: 732 445 5735; Email: ebright{at}waksman.rutgers.edu
Correspondence may also be addressed to Danny Reinberg. Tel: 212 263 9036; Fax: 212 263 9040; Email: reinbd01{at}med.nyu.edu
Received August 14, 2008. Revised September 4, 2008. Accepted September 9, 2008.
We have developed an approach that enables nonradioactive, ultrasensitive (attamole sensitivity) site-specific protein–protein photocrosslinking, and we have applied the approach to the analysis of interactions of 
-helix 2 (H2) of human TATA-element binding protein (TBP) with general transcription factor TFIIA and transcriptional repressor NC2. We have found that TBP H2 can be crosslinked to TFIIA in the TFIIA–TBP–DNA complex and in higher order transcription–initiation complexes, and we have mapped the crosslink to the ‘connector’ region of the TFIIA /β subunit (TFIIA/β). We further have found that TBP H2 can be crosslinked to NC2 in the NC2–TBP–DNA complex, and we have mapped the crosslink to the C-terminal ‘tail’ of the NC2 -subunit (NC2). Interactions of TBP H2 with the TFIIA/β connector and the NC2 C-terminal tail were not observed in crystal structures of TFIIA–TBP–DNA and NC2–TBP–DNA complexes, since relevant segments of TFIIA and NC2 were not present in truncated TFIIA and NC2 derivatives used for crystallization. We propose that interactions of TBP H2 with the TFIIA/β connector and the NC2 C-terminal tail provide an explanation for genetic results suggesting importance of TBP H2 in TBP–TFIIA interactions and TBP–NC2 interactions, and provide an explanation—steric exclusion—for competition between TFIIA and NC2.
Present addresses: Younggyu Kim, Department of Chemistry and Biochemistry, University of California, Los Angeles, CA 90095, USA. Adam Goodman, Northwest Biomedical Research and Consulting, West Linn, OR 97068, USA.
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors.