Nonradioactive, ultrasensitive site-specific protein–protein photocrosslinking: interactions of α-helix 2 of TATA-binding protein with general transcription factor TFIIA and transcriptional repressor NC2

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 α-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 α/β subunit (TFIIAα/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 α-subunit (NC2α). Interactions of TBP H2 with the TFIIAα/β connector and the NC2α 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 TFIIAα/β connector and the NC2α 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.